Multivalent metal salts of boronic acids

ABSTRACT

Salts of a pharmaceutically acceptable divalent metal and an organoboronic acid drug. Examples of such metals are calcium, magnesium and zinc. The organoboronic acid drug may be a boropeptide protease inhibitor. The salts may be formulated in oral dosage form.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.11/438,823, filed May 22, 2006, which is incorporated herein byreference, which is a divisional of U.S. application Ser. No.10/659,179, filed Sep. 9, 2003, which is incorporated herein byreference, which claims priority to GB 0220764.5, filed Sep. 9, 2002, GB0220822.1, filed Sep. 9, 2002, GB 0307817.7, filed Apr. 4, 2003, GB0311237.2, filed May 16, 2003, and GB 0315691.6, filed Jul. 4, 2003, allof which are hereby incorporated by reference.

BACKGROUND

The present disclosure relates to pharmaceutically useful productsobtainable from organoboronic acids. The disclosure also relates to theuse of members of the aforesaid class of products, to their formulation,their preparation, their synthetic intermediates and to other subjectmatter.

The disclosure further relates to oral pharmaceutical formulationscontaining the described products.

Boronic Acid Compounds

It has been known for some years that boronic acid compounds and theirderivatives, e.g. esters, have biological activities, notably asinhibitors or substrates of proteases. For example, Koehler et al.Biochemistry 10:2477, 1971 report that 2-phenylethane boronic acidinhibits the serine protease chymotrypsin at millimolar levels. Theinhibition of chymotrypsin and subtilisin by arylboronic acids(phenylboronic acid, m-nitro-phenylboronic acid, m-aminophenylboronicacid, m-bromophenylboronic acid) is reported by Phillip et al, Proc.Nat. Acad. Sci. USA 68:478-480, 1971. A study of the inhibition ofsubtilisin Carlsberg by a variety of boronic acids, especially phenylboronic acids substituted by Cl, Br, CH₃, H₂N, MeO and others, isdescribed by Seufer-Wasserthal et al, Biorg. Med. Chem. 2(1):35-48,1994.

In describing inhibitors or substrates of proteases, P1, P2, P3, etc.designate substrate or inhibitor residues which are amino-terminal tothe scissile peptide bond, and S1, S2, S3, etc., designate thecorresponding subsites of the cognate protease in accordance with:Schechter, I. and Berger, A. On the Size of the Active Site inProteases, Biochem. Biophys. Res. Comm., 27:157-162, 1967. In thrombin,the S1 binding site or “specificity pocket” is a well defined slit inthe enzyme, whilst the S2 and S3 binding subsites (also respectivelycalled the proximal and distal hydrophobic pockets) are hydrophobic andinteract strongly with, respectively, Pro and (R)-Phe, amongst others.

Pharmaceutical research into serine protease inhibitors has moved fromthe simple arylboronic acids to boropeptides, i.e. peptides containing aboronic acid analogue of an a-amino carboxylic acid. The boronic acidmay be derivatised, often to form an ester. Shenvi (EP-A-145441 and U.S.Pat. No. 4,499,082) disclosed that peptides containing an α-aminoboronicacid with a neutral side chain were effective inhibitors of elastase andhas been followed by numerous patent publications relating toboropeptide inhibitors of serine proteases. Specific, tight bindingboronic acid inhibitors have been reported for elastase (K_(i), 0.25nM), chymotrypsin (K_(i), 0.25 nM), cathepsin G (K_(i), 21 nM), α-lyticprotease (K_(i), 0.25 nM), dipeptidyl aminopeptidase type IV (K_(i), 16pM) and more recently thrombin (Ac-D-Phe-Pro-boroArg-OH (DuP 714 initialK_(i) 1.2 nM).

Claeson et al (U.S. Pat. No. 5,574,014 and others) and Kakkar et al (WO92/07869 and family members including U.S. Pat. No. 5,648,338) disclosethrombin inhibitors having a neutral C-terminal side chain, for examplean alkyl or alkoxyalkyl side chain.

Modifications of the compounds described by Kakkar et al are included inWO 96/25427, directed to peptidyl serine protease inhibitors in whichthe P2-P1 natural peptide linkage is replaced by another linkage. Asexamples of non-natural peptide linkages may be mentioned —CO₂—, —CH₂O—,—NHCO—, —CHYCH₂—, —CH═CH—, —CO(CH₂)_(p)CO— where p is 1, 2 or 3,—COCHY—, —CO₂—CH₂NH—, —CHY—NX—, —N(X)CH₂—N(X)CO—, —CH═C(CN)CO—,—CH(OH)—NH—, —CH(CN)—NH—, —CH(OH)—CH₂— or —NH— CHOH—, where X is H or anamino protecting group and Y is H or halogen, especially F. Particularnon-natural peptide linkages are —CO₂— or —CH₂O—.

Metternich (EP 471651 and U.S. Pat. No. 5,288,707, the latter beingassigned to Trigen Limited) discloses variants of Phe-Pro-BoroArgboropeptides in which the P3 Phe is replaced by an unnatural hydrophobicamino acid such as trimethylsilylalanine,p-tert.butyl-diphenyl-silyloxymethyl-phenylalanine orp-hydroxymethylphenylalanine and the P1 side chain may be neutral(alkoxyalkyl, alkylthioalkyl or trimethylsilylalkyl).

The replacement of the P2 Pro residue of borotripeptide thrombininhibitors by an N-substituted glycine is described in Fevig J M et alBioorg. Med. Chem. 8: 301-306 and Rupin A et al Thromb. Haemost.78(4):1221-1227, 1997. See also U.S. Pat. No. 5,585,360 (de Nanteuil etal).

Amparo (WO 96/20698 and family members including U.S. Pat. No.5,698,538) discloses peptidomimetics of the structureAryl-linker-Boro(Aa), where Boro(Aa) may be an aminoboronate residuewith a non-basic side chain, for example BoroMpg. The linker is of theformula —(CH₂)_(m)CONR— (where m is 0 to 8 and R is H or certain organicgroups) or analogues thereof in which the peptide linkage —CONR— isreplaced by —CSNR—, —SO₂NR—, —CO₂—, —C(S)O— or —SO₂O —. Aryl is phenyl,naphthyl or biphenyl substituted by one, two or three moieties selectedfrom a specified group. Most typically these compounds are of thestructure Aryl-(CH₂)_(n)—CONH—CHR²—BY¹Y², where R² is for example aneutral side chain as described above and n is 0 or 1.

Non-peptide boronates have been proposed as inhibitors of proteolyticenzymes in detergent compositions. WO 92/19707 and WO 95/12655 reportthat arylboronates can be used as inhibitors of proteolytic enzymes indetergent compositions. WO 92/19707 discloses compounds substituted metato the boronate group by a hydrogen bonding group, especially acetamido(—NHCOCH₃), sufonamido (—NHSO₂CH₃) and alkylamino. WO 95/12655 teachesthat ortho-substituted compounds are superior.

Boronate enzyme inhibitors have wide application, from detergents tobacterial sporulation inhibitors to pharmaceuticals. In thepharmaceutical field, there is patent literature describing boronateinhibitors of serine proteases, for example thrombin, factor Xa,kallikrein, elastase, plasmin as well as other serine proteases likeprolyl endopeptidase and Ig AI Protease. Thrombin is the last proteasein the coagulation pathway and acts to hydrolyse four small peptidesform each molecule of fibrinogen, thus deprotecting its polymerisationsites. Once formed, the linear fibrin polymers may be cross-linked byfactor XIIIa, which is itself activated by thrombin. In addition,thrombin is a potent activator of platelets, upon which it acts atspecific receptors. Thrombin also potentiates its own production by theactivation of factors V and VIII.

Other aminoboronate or peptidoboronate inhibitors or substrates ofserine proteases are described in:

-   -   U.S. Pat. No. 4,935,493    -   EP 341661    -   WO 94/25049    -   WO 95/09859    -   WO 96/12499    -   WO 96/20689    -   Lee S-L et al, Biochemistry 36:13180-13186, 1997    -   Dominguez C et al, Bioorg. Med. Chem. Lett. 7:79-84, 1997    -   EP 471651    -   WO 94/20526    -   WO 95/20603    -   WO97/05161    -   U.S. Pat. No. 4,450,105    -   U.S. Pat. No. 5,106,948    -   U.S. Pat. No. 5,169,841.

Peptide boronic acid inhibitors of hepatic C virus protease aredescribed in WO 01/02424.

Matteson D S Chem. Rev. 89: 1535-1551, 1989 reviews the use of α-haloboronic esters as intermediates for the synthesis of inter alia aminoboronic acids and their derivatives. Matteson describes the use ofpinacol boronic esters in non-chiral synthesis and the use of pinanediolboronic esters for chiral control, including in the synthesis of aminoand amido boronate esters.

Contreras et al J. Organomet. Chem. 246: 213-217, 1983 describe howintramolecular N→B coordination was demonstrated by spectroscopicstudies on cyclic boronic esters prepared by reacting Me₂CHCMe₂—BH₂ withdiethanolamines.

Boronic acid and ester compounds have displayed promise as inhibitors ofthe proteasome, a multicatalytic protease responsible for the majorityof intracellular protein turnover. Ciechanover, Cell, 79:13-21, 1994,teaches that the proteasome is the proteolytic component of theubiquitin-proteasome pathway, in which proteins are targeted fordegradation by conjugation to multiple molecules of ubiquitin.Ciechanover also teaches that the ubiquitin-proteasome pathway plays akey role in a variety of important physiological processes.

Adams et al, U.S. Pat. No. 5,780,454 (1998), U.S. Pat. No. 6,066,730(2000), U.S. Pat. No. 6,083,903 (2000) and equivalent WO 96/13266, andU.S. Pat. No. 6,297,217 (2001) describe peptide boronic ester and acidcompounds useful as proteasome inhibitors. These documents also describethe use of boronic ester and acid compounds to reduce the rate of muscleprotein degradation, to reduce the activity of NF-κB in a cell, toreduce the rate of degradation of p53 protein in a cell, to inhibitcyclin degradation in a cell, to inhibit the growth of a cancer cell, toinhibit antigen presentation in a cell, to inhibit NF-κB dependent celladhesion, and to inhibit HIV replication. Brand et al, WO 98/35691,teaches that proteasome inhibitors, including boronic acid compounds,are useful for treating infarcts such as occur during stroke ormyocardial infarction. Elliott et al, WO 99/15183, teaches thatproteasome inhibitors are useful for treating inflammatory andautoimmune diseases.

Unfortunately, organoboronic acids can be relatively difficult to obtainin analytically pure form. Thus, alkylboronic acids and their boroxinesare often air-sensitive. Korcek et al, J. Chem. Soc. Perkin Trans.2:242, 1972, teaches that butylboronic acid is readily oxidized by airto generate 1-butanol and boric acid.

It is known that derivatisation of boronic acids as cyclic estersprovides oxidation resistance. For example, Martichonok V et al J. Am.Chem. Soc. 118: 950-958, 1996 state that diethanolamine derivatisationprovides protection against possible boronic acid oxidation. U.S. Pat.No. 5,681,978 (Matteson D S et al) teaches that 1,2-diols and 1,3 diols,for example pinacol, form stable cyclic boronic esters that are noteasily oxidised.

Wu et al, J. Pharm. Sci., 89:758-765, 2000, discuss the stability of thecompound N-(2-pyrazine) carbonyl-phenylalanine-leucine boronic acid(LDP-341, also known as bortezomib), an anti-cancer agent. It isdescribed how “during an effort to formulate [LDP-341] for parenteraladministration, the compound showed erratic stability behaviour”. Thedegradation pathways were investigated and it was concluded that thedegradation was oxidative, the initial oxidation being attributed toperoxides or molecular oxygen and its radicals.

WO 02/059131 discloses boronic acid products which are described asstable. In particular, these products are certain boropeptides and/orboropeptidomimetics in which the boronic acid group has been derivatisedwith a sugar. The disclosed sugar derivatives, which have hydrophobicamino acid side chains, are of the formula

wherein:

P is hydrogen or an amino-group protecting moiety;

R is hydrogen or alkyl;

A is 0, 1 or 2;

R¹, R² and R³ are independently hydrogen, alkyl, cycloalkyl, aryl or—CH₂—R⁵;

R⁵, in each instance, is one of aryl, aralkyl, alkaryl, cycloalkyl,heterocyclyl, heteroaryl, or —W—R⁶, where W is a chalcogen and R⁶ isalkyl;

where the ring portion of any of said aryl, aralkyl, alkaryl,cycloalkyl, heterocyclyl, or heteroaryl in R¹, R², R³ or R⁵ can beoptionally substituted; and

Z¹ and Z² together form a moiety derived from a sugar, wherein the atomattached to boron in each case is an oxygen atom.

Some of the disclosed compounds are sugar derivatives of LDP-341 (seeabove).

Many drugs comprise an active moiety which is a carboxylic acid. Thereare a number of differences between carboxylic acids and boronic acids,whose effects on drug delivery, stability and transport (amongst others)have not been investigated. One feature of trivalent boron compounds isthat the boron atom is sp² hybridised, which leaves an empty 2p_(z)orbital on the boron atom. A molecule of the type BX₃ can therefore actas an electron-pair acceptor, or Lewis acid. It can use the empty 2p_(z)orbital to pick up a pair of nonbonding electrons from a Lewis base toform a covalent bond. BF₃ therefore reacts with Lewis bases such as NH₃to form acid-base complexes in which all of the atoms have a filledshell of valence electrons.

Boric acid, accordingly, can act as a Lewis acid, accepting OH⁻:

B(OH)₃+H₂O→B(OH)₄ ⁻+H⁺

Further, boronic acids of the type RB(OH)₂ are dibasic and have twopKa's. Another point of distinction about boron compounds is theunusually short length of bonds to boron, for which three factors may beresponsible:

1. Formation of pn-pn bonds;

2. Ionic-covalent resonance;

3. Reduced repulsions between non-bonding electrons.

The presumed equilibria of boronic and carboxylic acids in aqueous KOHare shown below (excluding formation of RBO₂ ²⁻):

Thrombosis

Hemostasis is the normal physiological condition of blood in which itscomponents exist in dynamic equilibrium. When the equilibrium isdisturbed, for instance following injury to a blood vessel, certainbiochemical pathways are triggered leading, in this example, to arrestof bleeding via clot formation (coagulation). Coagulation is a dynamicand complex process in which proteolytic enzymes such as thrombin play akey role. Blood coagulation may occur through either of two cascades ofzymogen activations, the extrinsic and intrinsic pathways of thecoagulation cascade. Factor VIIa in the extrinsic pathway, and FactorIXa in the intrinsic pathway are important determinants of theactivation of factor X to factor Xa, which itself catalyzes theactivation of prothrombin to thrombin, whilst thrombin in turn catalysesthe polymerization of fibrinogen monomers to fibrin polymer. The lastprotease in each pathway is therefore thrombin, which acts to hydrolyzefour small peptides (two FpA and two FpB) from each molecule offibrinogen, thus deprotecting its polymerization sites. Once formed, thelinear fibrin polymers may be cross-linked by factor XIIIa, which isitself activated by thrombin. In addition, thrombin is a potentactivator of platelets, upon which it acts at specific receptors.Thrombin activation of platelets leads to aggregation of the cells andsecretion of additional factors that further accelerate the creation ofa hemostatic plug. Thrombin also potentiates its own production by theactivation of factors V and VIII (see Hemker and Beguin in: Jolles, et.al., “Biology and Pathology of Platelet Vessel Wall Interactions,” pp.219-26 (1986), Crawford and Scrutton in: Bloom and Thomas, “Haemostasisand Thrombosis,” pp. 47-77, (1987), Bevers, et. al., Eur. J. Biochem.122:429-36, 1982, Mann, Trends Biochem. Sci 12:229-33, 1987).

Proteases are enzymes which cleave proteins at specific peptide bonds.Cuypers et al., J. Biol. Chem. 257:7086, 1982, and the references citedtherein, classify proteases on a mechanistic basis into five classes:serine, cysteinyl or thiol, acid or aspartyl, threonine andmetalloproteases. Members of each class catalyse the hydrolysis ofpeptide bonds by a similar mechanism, have similar active site aminoacid residues and are susceptible to class-specific inhibitors. Forexample, all serine proteases that have been characterised have anactive site serine residue.

The coagulation proteases thrombin, factor Xa, factor VIIa, and factorIXa are serine proteases having trypsin-like specificity for thecleavage of sequence-specific Arg-Xxx peptide bonds. As with otherserine proteases, the cleavage event begins with an attack of the activesite serine on the scissile bond of the substrate, resulting in theformation of a tetrahedral intermediate. This is followed by collapse ofthe tetrahedral intermediate to form an acyl enzyme and release of theamino terminus of the cleaved sequence. Hydrolysis of the acyl enzymethen releases the carboxy terminus.

As indicated above, platelets play two important roles in normalhemostasis. First, by aggregating, they constitute the initialhemostatic plug which immediately curtails bleeding from broken bloodvessels. Secondly, the platelet surface can become activated andpotentiate blood clotting, a property referred to as plateletprocoagulant activity. This may be observed as an increase in the rateof activation of prothrombin by factor Xa in the presence of factor Vaand Ca²⁺, referred to as the prothrombinase reaction. Normally, thereare few (if any) clotting factors on the surface of unstimulatedplatelets but, when platelets are activated, negatively chargedphospholipids (phosphatidylserine and phospatidylinositol) that arenormally on the cytoplasmic side of the membrane become available andprovide a surface on which two steps of the coagulation sequence occur.The phospholipid on the surface of activated platelets profoundlyaccelerates the reactions leading to the formation of thrombin, so thatthrombin can be generated at a rate faster than its neutralisation byantithrombin III. The reactions that occur on the platelet surfaces arenot easily inhibited by the natural anticoagulants in blood such asantithrombin III, either with or without heparin. (See Kelton and Hirschin: Bloom and Thomas, “Haemostasis and Thrombosis,” pp. 737-760, (1981);Mustard et al in: Bloom and Thomas, “Haemostasis and Thrombosis,” pp.503526, (1981); Goodwin et al; Biochem. J. 308:15-21, 1995).

A thrombus can be considered as an abnormal product of a normalmechanism and can be defined as a mass or deposit formed from bloodconstituents on a surface of the cardiovascular system, for example ofthe heart or a blood vessel. Thrombosis can be regarded as thepathological condition wherein improper activity of the hemostaticmechanism results in intravascular thrombus formation. Three basic typesof thrombi are recognised:

-   -   the white thrombus which is usually seen in arteries and        consists chiefly of platelets;    -   the red thrombus which is found in veins and is composed        predominantly of fibrin and red cells;    -   the mixed thrombus which is composed of components of both white        and red thrombi.

The composition of thrombi is influenced by the velocity of blood flowat their sites of formation. In general white platelet-rich thrombi formin high flow systems, while red coagulation thrombi form in regions ofstasis. The high shear rate in arteries prevents the accumulation ofcoagulation intermediates on the arterial side of the circulation: onlyplatelets have the capacity to form thrombi binding to the area ofdamage via von Willebrand factor. Such thrombi composed only ofplatelets are not stable and disperse. If the stimulus is strong thenthe thrombi will form again and then disperse continually until thestimulus has diminished. For the thrombus to stabilise, fibrin mustform. In this respect, small amounts of thrombin can accumulate withinthe platelet thrombus and activate factor Va and stimulate the plateletprocoagulant activity. These two events lead to an overall increase inthe rate of activation of prothrombin by factor Xa of 300,000 fold.Fibrin deposition stabilises the platelet thrombus. Indirect thrombininhibitors, for example heparin, are not clinically effective atinhibiting stimulation of platelet procoagulant activity. Accordingly, atherapeutic agent which inhibits platelet procoagulant activity would beuseful for treating or preventing arterial thrombotic conditions.

On the venous side of circulation, the thrombus is comprised of fibrin:thrombin can accumulate because of the slower flow on the venous sideand platelets play only a minor role.

Thrombosis is thus not considered to be a single indication but, rather,is a class of indications embracing distinct sub-classes for whichdiffering therapeutic agents and/or protocols may be appropriate. Thus,regulatory authorities treat disorders such as, for example, deep veinthrombosis, cerebrovascular arterial thrombosis and pulmonary embolismas distinct indications for the purposes of licensing medicines. Twomain sub-classes of thrombosis are arterial thrombosis and venousthrombosis. Arterial thrombosis includes such specific disorders asacute coronary syndromes [for example acute myocardial infarction (heartattack, caused by thrombosis in a coronary artery)], cerebrovasculararterial thrombosis (stroke, caused by thrombosis in the cerebrovasculararterial system) and peripheral arterial thrombosis. Examples ofconditions caused by venous thrombosis are deep vein thrombosis andpulmonary embolism.

The management of thrombosis commonly involves the use of antiplateletdrugs (inhibitors of platelet aggregation) to control futurethrombogenesis and thrombolytic agents to lyse the newly formed clot,either or both such agents being used in conjunction or combination withanticoagulants. Anticoagulants are used also preventatively(prophylactically) in the treatment of patients thought susceptible tothrombosis.

Currently, two of the most effective classes of drugs in clinical use asanticoagulants are the heparins and the vitamin K antagonists. Theheparins are ill-defined mixtures of sulfated polysaccharides that bindto, and thus potentiate, the action of antithrombin III. AntithrombinIII is a naturally occurring inhibitor of the activated clotting factorsIXa, Xa, XIa, thrombin and probably XIIa (see Jaques, Pharmacol. Rev.31:99-166, 1980). The vitamin K antagonists, of which warfarin is themost well-known example, act indirectly by inhibiting the post-ribosomalcarboxylations of the vitamin K dependent coagulation factors II, VII,IX and X (see Hirsch, Semin. Thromb. Hemostasis 12:1-11, 1986). Whileeffective therapies for the treatment of thrombosis, heparins andvitamin K antagonists have the unfortunate side effects of bleeding,heparin-induced thrombocytopenia (in the case of heparin) and markedinterpatient variability, resulting in a small and unpredictabletherapeutic safety margin.

The use of direct acting inhibitors of thrombin and other serineprotease enzymes of the coagulation system is expected to alleviatethese problems. To that end, a wide variety of serine proteaseinhibitors have been tested, including boropeptides, i.e. peptidescontaining a boronic acid analogue of an a-amino acid. Whilst directacting boronic acid thrombin inhibitors have been discussed earlier inthis specification, they are further described in the following section.

Neutral P1 Residue Boropeptide Thrombin Inhibitors

Claeson et al (U.S. Pat. No. 5,574,014 and others) and Kakkar et al (WO92/07869 and family members including U.S. Pat. No. 5,648,338) discloselipophilic thrombin inhibitors having a neutral (uncharged) C-terminal(P1) side chain, for example an alkoxyalkyl side chain.

The Claeson et al and Kakkar et al patent families disclose boronateesters containing the amino acid sequence D-Phe-Pro-BoroMpg[(R)-Phe-Pro-BoroMpg], which are highly specific inhibitors of thrombin.Of these compounds may be mentioned in particularCbz-(R)-Phe-Pro-BoroMpg-OPinacol (also known as TRI 50b). Thecorresponding free boronic acid is known as TRI 50c. For furtherinformation relating to TRI 50b and related compounds, the reader isreferred to the following documents:

-   -   Elgendy S et al., in The Design of Synthetic Inhibitors of        Thrombin, Claeson G et al Eds, Advances in Experimental        Medicine, 340:173-178, 1993.    -   Claeson G et al, Biochem J. 290:309-312, 1993    -   Tapparelli C et al, J Biol Chem, 268:4734-4741, 1993    -   Claeson G. in The Design of Synthetic Inhibitors of Thrombin,        Claeson G et al Eds, Advances in Experimental Medicine,        340:83-91, 1993    -   Phillip et al, in The Design of Synthetic Inhibitors of        Thrombin, Claeson G et al Eds, Advances in Experimental        Medicine, 340:67-77, 1993    -   Tapparelli C et al, Trends Pharmacol. Sci. 14:366-376, 1993    -   Claeson G, Blood Coagulation and Fibrinolysis 5:411-436, 1994    -   Elgendy et al, Tetrahedron 50:3803-3812, 1994    -   Deadman 3 et al, J. Enzyme Inhibition 9:29-41, 1995    -   Deadman 3 et al, J. Medicinal Chemistry 38:1511-1522, 1995.

The tripeptide sequence of TRI 50b has three chiral centres. The Pheresidue is considered to be of (R)-configuration and the Pro residue ofnatural (S)-configuration, at least in compounds with commerciallyuseful inhibitor activity; the Mpg residue is believed to be of(R)-configuration in isomers with commercially useful inhibitoractivity. Thus, the active, or most active, TRI 50b stereoisomer isconsidered to be of R,S,R configuration and may be represented as:

-   -   (RSR)-TRI 50b: Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg-Pinacol

Whilst indirect acting thrombin inhibitors have been found useful forthe treatment of patients susceptible to or suffering from venousthrombosis, the same is not true of arterial thrombosis, because itwould be necessary to raise the dosage used in the treatment of venousthrombosis by many times in order to treat (prevent) arterialthrombosis. Such raised dosages typically cause bleeding, which makesindirect acting thrombin inhibitors unsuitable or less preferable fortreating arterial thrombosis. Heparin and its low molecular weightderivatives are indirect thrombin inhibitors, and so are unsuitable totreat arterial thrombosis. Oral direct thrombin inhibitors are indevelopment for arterial indications but may have lower than desirabletherapeutic indices, i.e. may have higher than desirable levels ofbleeding at therapeutic doses.

Oral Absorption

Absorption in the gastrointestinal tract can be by an active or apassive route. Active absorption by transport mechanisms tends to bevariable between individuals and with intestinal content (Gustafsson etal, Thrombosis Research, 101:171-181, 2001). The upper intestine hasbeen identified as the principal site of oral drug absorption. Inparticular, the duodenum is the customary target site for absorption oforally administered drugs because of its large surface area. Theintestinal mucosa acts as a barrier that controls passive transcellularabsorption: the absorption of ionic species is blocked whilst thetranscellular absorption of lipophilic molecules is favoured (Palm K etal., J. Pharmacol and Exp. Therapeutics, 291:435-443, 1999).

Orally administered drugs are required to be consistently and adequatelyabsorbed. Variability of absorption between individuals or betweendifferent occasions in the same individual is unwelcome. Similarly,drugs which have a low level of bioavailability (only a small portion ofthe administered active agent is absorbed) are generally unacceptable.

Non-ionised compounds are favoured for passive absorption, a routeassociated with invariability, and are therefore preferred forconsistent absorption. Lipophilic species are particularly favoured bypassive absorption mechanisms and, accordingly, non-ionic, lipophilicdrugs are indicated to be most favoured for consistent and high oralabsorption.

Many organoboronic acid compounds may be classified as lipophilic orhydrophobic. Typically, such compounds include amongst others:

-   -   boropeptides of which all or a majority of the amino acids are        hydrophobic    -   boropeptides of which at least half of the amino acids are        hydrophobic and which have a hydrophobic N-terminal substituent        (amino protecting group)    -   non-peptides based on hydrophobic moieties.

Typical functionalities required for interaction of drugs with theirphysiological targets are functional groups such as carboxylic andsulphonic acids. These groups exist as the protonated form in thestomach (at pH 2-3), but will be ionised to some extent at the higher pHof the intestinal fluid. One strategy that has been used to avoid theionisation of the carboxylates or sulphonates is to present them asester forms, which are cleaved once absorbed into the vascular lumen.

For example, the direct acting thrombin inhibitor melagatran, which hassub-optimal gastrointestinal absorption, has terminal carboxy andamidino groups and is a pure zwitterion at pH 8-10 when the carboxylicacid and amidino groups are both charged. A prodrug H 376/95 wastherefore developed which has protecting groups for the carboxylic acidand for the amidine and is a more lipophilic molecule than melagatran.The prodrug has a permeability coefficient across cultured epithelialCaco-2 cells 80 times higher than that of melagatran and oralbioavailability 2.7-5.5 times higher than that of melagatran as well asmuch smaller variability in the area under the drug plasma concentrationvs. time curve (Gustafsson et al, Thrombosis Research, 101:171-181,2001).

Boronic acids are divalent functional groups, with boron-oxygen bondlengths (1.6 Å) more typical of single bonds, unlike superficiallycomparable C—O and S—O bonds in carboxylic and sulphonic acids.Consequently the boronic acid group has two ionisation potentials. Theboronic acid group will be partly ionised at pH's of the duodenal fluidand not suited to the desired passive duodenal uptake. Thus, a chargedboronate inhibitor H-D-PheProBoroArg is absorbed by a predominantlyactive transport mechanism (Saitoh, H. and Aungst, B. J., Pharm. Res.,16:1786-1789, 1999).

Oral Absorption of Boropeptides, Boropeptidomimetics and otherOrganoboronates

The boronate ester group of TRI 50b is rapidly cleaved in the conditionsof the plasma to form the corresponding boronic acid group, which isconsidered to be the active moiety which inhibits the catalytic site ofthrombin.

The peptide boronic acid formed by such cleavage of TRI 50b (the acid isdesignated TRI 50c) is relatively insoluble in water, especially atacidic or neutral pH, and tends to be poorly absorbed in the stomach andduodenum. The acid has the structure Cbz-Phe-Pro-BoroMpg-OH.

Whereas the peptide boronic acid Cbz-Phe-Pro-BoroMpg-OH is partlyionised under duodenal conditions and, to that extent, unfavoured forpassive transport, esters of the acid are designed for a high rate ofpassive (thus consistent) transport. The tripeptide sequence Phe-Pro-Mpghas a non-basic P1 side chain (specifically, methoxypropyl), such thatthe tripeptide consists of three non-polar amino acids. The esters ofthe peptide boronic acid are non-ionisable and the ester-forming speciesfurther impart lipophilic properties, so encouraging a high rate ofpassive transport.

Computational techniques have confirmed that TRI 50b and other diolesters of Cbz-Phe-Pro-BoroMpg-OH can be predicted to have goodbioavailability. Thus, polar surface area (PSAd) is a parameterpredictive of bioavailability and PSAd values of greater than 60 Åcorrelate well with passive transcellular transport and withbioavailability of known drugs (Kelder, J. Pharm. Res., 16:1514-1519,1999). Measurements for diol esters of the above peptide boronic acid,including the pinacol ester TRI 50b, show that the diol esters have PSAdvalues well above 60 Å, predictive of passive transport and goodbioavailability as shown in Table 1:

TABLE 1 PSAd values of selected diol esters of Cbz-Phe-Pro-BoroMpg-OHDiol PSAd Value Pinacol 98.74 Pinanediol 90.64

The corresponding monohydroxy alcohol (e.g. alkanol) esters wereconsidered too unstable, spontaneously cleaving to liberate the acidin-vitro. Esters of diols such as pinanediol and pinacol have enhancedkinetic stability over esters of monohydroxy alcohols, in that afterpartial hydrolysis to the mono-ester derivative they will tend toreassociate by a facile intra-molecular reaction.

BRIEF SUMMARY OF THE DISCLOSURE

To counterbalance the highly desirable features of TRI 50b, it has beendiscovered that TRI 50b tends to hydrolyse. Thus in acid conditions, forexample HPLC assay, TRI 50b is converted to the acid form with a shorthalf life, which implies potential hydrolysis in the duodenum andelsewhere in the gastro-intestinal tract into ionic species which wouldresist passive transport and, if anything, be absorbed by activetransport, indicative at best of variable bioavailability.

The instability of TRI 50b to hydrolysis also presents potentialdisadvantages in preparation of the compound and its formulation, aswell as in the storage of pharmaceutical formulations containing it.

Another challenging difficulty which has been posed by TRI 50b is thatthe data show significant variation in bioavailability between subjects.Such variability can make a drug candidate unacceptable and it wouldtherefore be desirable to reduce the observed variability.

An ideal solution to the instability of TRI 50b would be development ofa diol ester more stable to hydrolysis, as such a diol ester like TRI50b can be predicted to be oxidation resistant as compared with TRI 50c.In this regard, it is known that ring size can affect boronate stabilityand glycolato boron has been shown to have enhanced aqueous stabilitycompared to pinacol (D. S. Matteson, Stereodirected Synthesis withOrganoboranes, Springer-Verlag, 1995, ch.1). Similarly, the pinanediolester is more stable than the pinacol; this is believed to be becausethe pinanediol group is highly sterically hindered and disfavoursnucleophilic attack on the boron. In fact transesterification frompinacol to pinanediol has been reported (Brosz, C S, Tet. Assym,8:1435-1440, 1997) whereas the reverse process is unfavourable. Thepinanediol ester however is considered too slow to cleave in plasma andthere remains a need to provide an improved diol ester.

Another solution to the instability of TRI 50b would be to administer inits place TRI 50c. However, TRI 50c data suggest that TRI 50c toosuffers from variability in bioavailability.

TRI 50c suffers further from instability, in that there is a problematictendency for the boropeptide moiety itself to degrade via de-boronation(carbon-boron bond cleavage), by a pathway previously considered to beoxidative as taught by the literature teaching (e.g. Wu et al, discussedabove). The level of degradation can be remarkably high.

The properties discussed above of TRI 50b and TRI 50c will not berestricted to such compounds but will be shared by other boropeptideesters and acids, even if the properties of such other boropeptidesdiffer quantitatively.

The present disclosure is predicated on, amongst other things, thefinding that certain organoboronic acid products are indicated to be ofenhanced stability. Another basis of the disclosure is the provision oforganoboronic acid products having unexpectedly favourablebioavailability.

The benefits of the present disclosure include a solution to the problemof boronate diol ester and especially TRI 50b instability, that is tosay the presently disclosed products provide inter aliapharmacologically active compounds which are more stable than TRI 50band other comparable esters in the sense of stability to hydrolysis. Thedisclosure further includes a solution to the problem of organoboronicacid instability, that is to say the presently disclosed productsprovide inter alia pharmacologically active compounds which are morestable to deboronation than TRI 50c. The stability provided within theframework of the disclosure is not absolute but is improved relative tothe comparator compounds. The benefits offered by the disclosure furtherinclude the provision of unexpected products which, contrary toexpectation, have a particularly low variability in oralbioavailability.

In one aspect, disclosed herein is a salt of a pharmaceuticallyacceptable multivalent (at least divalent) metal and an organoboronicacid drug. As a class, such salts are not only contrary to the directionof the prior art but additionally have an improved level of stabilitywhich cannot be explained or predicted on the basis of known chemistry.The salts are indicated to have unexpectedly high and consistent oralbioavailability not susceptible of explanation on the basis of knownmechanisms.

The disclosure includes amongst other things a class of salts of whichthe drug (the free acid) has no charged group at physiological pH otherthan its boronate (boronic acid) moiety and an amino moiety. Thedisclosure includes a class of salts of which the drug (the free acid)is a peptide boronate all of whose amino acid residues have unchargedside chains.

In certain embodiments the organoboronic acid is hydrophobic. Particularorganoboronic acids have a partition coefficient between 1-n-octanol andwater expressed as log P of greater than 1.0 at physiological pH and 25°C. Some useful hydrophobic organoboronic acids have a partitioncoefficient of at least 1.5. A class of useful hydrophobic organoboronicacids has a partition coefficient of no more than 5.

One particular class of salts comprises those wherein the organoboronicacid comprises a boropeptide or boropeptidomimetic. Boropeptide drugswhich may beneficially be prepared as salts include without limitationthose of the formula X-(aa)_(n)—B(OH)₂, where X is H or anamino-protecting group, n is 2, 3 or 4, (especially 2 or 3) and each aais independently a hydrophobic amino acid, whether natural or unnatural.

Disclosed as certain examples are multivalent metal salts of hydrophobicboronic acid inhibitors of thrombin. Such inhibitors may containhydrophobic amino acids, and this class of amino acids includes thosewhose side chain is hydrocarbyl, hydrocarbyl containing an in-chainoxygen and/or linked to the remainder of the molecule by an in-chainoxygen or heteroaryl, or any of the aforesaid groups when substituted byhydroxy, halogen or trifluoromethyl. Representative hydrophobic sidechains include alkyl, alkoxyalkyl, either of the aforesaid whensubstituted by at least one aryl or heteroaryl, aryl, heteroaryl, arylsubstituted by at least one alkyl and heteroaryl substituted by at leastone alkyl. Proline and other imino acids which are ring-substituted bynothing or by one of the moieties listed in the previous sentence arealso hydrophobic.

Some hydrophobic side chains contain from 1 to 20 carbon atoms, e.g.non-cyclic moieties having 1, 2, 3 or 4 carbon atoms. Side chainscomprising a cyclic group typically but not necessarily contain from 5to 13 ring members and in many cases are phenyl or alkyl substituted byone or two phenyls.

Included are inhibitors which contain hydrophobic non-peptide moieties,which are typically based on moieties which may form a side chain of ahydrophobic amino acid, as described above.

Hydrophobic compounds may contain, for example, one amino group and/orone acid group (e.g. —COOH, —B(OH)₂). Generally, they do not containmultiple polar groups of any one type.

One class of hydrophobic organoboronic acids have a partitioncoefficient between 1-n-octanol and water expressed as log P of greaterthan 1 at physiological pH and 25° C. For example, TRI 50c has apartition coefficient of approximately 2.

Some sub-classes of hydrophobic organoboronic acids are those describedby Formulae (I) and (III)) below, under the heading “DetailedDescription of Several Examples”.

The disclosure includes base addition salts of peptide boronic acidswhich have a partition coefficient between 1-n-octanol and waterexpressed as log P of greater than 1.0 at physiological pH and 25° C.Some useful peptide boronic acids have a partition coefficient of atleast 1.5. A class of useful hydrophobic peptide boronic acids has apartition coefficient of no more than 5.

In a sub-class of the salts of boropeptides/boropeptidomimetics, theorganoboronic acid is of the formula (I):

where:

R¹ is H or a neutral side group;

R² is H or C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen orsulfur and optionally substituted by a substituent selected from halo,hydroxy and trifluoromethyl;

-   -   or R¹ and R² together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur;

R³ is the same as or different from R¹ provided that no more than one ofR¹ and R² is H, and is H or a neutral side group;

R⁴ is H or C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen orsulfur and optionally substituted by a substituent selected from halo,hydroxy and trifluoromethyl;

-   -   or R³ and R⁴ together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur; and

R⁵ is X-E- wherein E is nothing or a hydrophobic moiety selected fromthe group consisting of amino acids (natural or unnatural) and peptidesof two or more amino acids (natural or unnatural) of which more thanhalf are hydrophobic and X is H or an amino-protecting group.

Also disclosed are pharmaceutically acceptable multivalent metal saltsof a peptide boronic acid of formulae (II), (III) or (IV) below.

The boronic acids of formulae (III) and (IV) inhibit thrombin. Theyexhibit anti-thrombotic activity in both venous and arterial contexts,and are considered to inhibit platelet pro-coagulant activity. Oneexample of a boronic acid of formulae (III) and (IV) is TRI 50c.

The Examples in this disclosure contain data showing that the calciumsalt of TRI 50c is markedly less soluble than the potassium salt and yethas higher oral bioavailability and higher consistency of oralbioavailability. The finding of an inverse relationship betweensolubility and bioavailability of two salts is particularlyunpredictable. There is no known property of organoboronic acid drugswhich accounts for this finding. The disclosure therefore includesamongst other subject matter a TRI 50c derivative which enhancesstability as compared with TRI 50b and reduces the variability inabsorption which has been observed with TRI 50b and TRI 50c, andadvantageously enables adequately consistent and high bioavailability.

The Examples in this disclosure also contain data demonstrating that thecalcium salt of TRI 50c is markedly more, stable than TRI 50c. Again,there is no known property which accounts for this finding.

The families of compounds represented by formulae (III) and (IV)represent near neighbours of TRI 50c which can be predicted to haveparticularly similar properties to TRI 50c.

Calcium is a representative of a class of pharmaceutically acceptablemultivalent metals. It is also a representative of a class ofpharmaceutically acceptable divalent metals; as other members of theclass may be mentioned magnesium and zinc.

TRI 50c is distinguished from most other organic acid drugs in that theacid group of TRI 50c is a boronic acid and not a carboxylic acid. Thedata in this disclosure are indicative of multivalent metal salts oforganoboronic acid drugs providing a technical effect, not linked tosolubility, which enhances the amount and consistency ofbioavailability. It does not follow that, because the effect is notlinked to solubility, there will in every individual case be for thatacid a quantitative relationship between solubility and bioavailabilitylike that observed for TRI 50c.

There is a debate in the literature as to whether boronates in aqueoussolution form the ‘trigonal’ B(OH)₂ or ‘tetrahedral’ B(OH)₃ ⁻ boronspecies, but NMR evidence seems to indicate that at a pH below the firstpKa of the boronic acid the main boron species is the neutral B(OH)₂. Inthe duodenum the pH is likely to be between 6 and 7, so the trigonalspecies is likely to be predominant here. In any event, the symbol—B(OH)₂ includes tetrahedral as well as trigonal boron species, andthroughout this specification symbols indicating trigonal boron speciesembrace also tetrahedral species. The symbol may further include boronicgroups in anhydride form.

The salts may be in the form of solvates, particularly hydrates.

The salts may comprise, or consist essentially of, acid salts in whichthe boronic acid is singly deprotonated. The disclosure thereforeincludes products having a metal/boronate stoichiometry consistent withthe boronate groups in the product predominantly (more than 50 mol %)carrying a single negative charge.

Oral formulations of the salts are also provided herein. In particular,there are provided oral formulations comprising the salts in the solidphase, for example particulate salts formulated as compressed tablets oras capsules.

According to a further aspect of the present disclosure, there isprovided a method of treatment of a condition where anti-thromboticactivity is required which method comprises oral administration of atherapeutically effective amount of a multivalent metal salt of aboronic acid of formula (III) to a person suffering from, or at risk ofsuffering from, such a condition.

The salts described herein include products obtainable by (having thecharacteristics of a product obtained by) reaction of the boronic acidwith a base of a multivalent metal and the term “salt” herein is to beunderstood accordingly. The term “salt” in relation to the disclosedproducts, therefore, does not necessarily imply that the productscontain discrete cations and anions and is to be understood as embracingproducts which are obtainable using a reaction of a boronic acid and abase. The disclosure embraces products which, to a greater or lesserextent, are in the form of a coordination compound. The disclosure thusprovides also products obtainable by (having the characteristics of aproduct obtained by) reaction of an organoboronic acid drug with amultivalent metal base a well as the therapeutic, includingprophylactic, use of such products.

The present disclosure is not limited as to the method of preparation ofthe salts, provided that they contain a boronate species derived fromboronic acid (I) and a counter-ion. Such boronate species may beboronate anions in any equilibrium form thereof. The term “equilibriumform” refers to differing forms of the same compounds which may berepresented in an equilibrium equation (e.g. boronic acid in equilibriumwith a boronic anhydride and in equilibrium with different boronateions). Boronates in the solid phase may form anhydrides and thedisclosed boronate salts when in the solid phase may comprise boronateanhydrides, as a boronic equilibrium species. It is not required thatthe salts be prepared by reaction of a base containing the counter-ionand the boronic acid (I). Further, the disclosure includes salt productswhich might be regarded as indirectly prepared by such an acid/basereaction as well as salts obtainable by (having the characteristics ofproducts obtained by) such indirect preparation. As examples of possiblyindirect preparation may be mentioned processes in which, after initialrecovery of the salt, it is purified and/or treated to modify itsphysicochemical properties, for example to modify solid form or hydrateform, or both.

In some embodiments the salts comprise anhydride species; in others theyare essentially free of anhydride species.

The salts may be in isolated form. The salts may have a purity, e.g. asdetermined by the method of Example 13, of at least about 90%, e.g. ofgreater than or equal to about 95%. In the case of pharmaceuticalformulations, such salt forms may be combined with pharmaceuticallyacceptable diluents, excipients or carriers.

The disclosure includes a method for preparing the salts from thecorresponding boronic acid as an intermediate, as well as theintermediate boronic acid of Formula (III) and a method for preparingit.

Further aspects and embodiments of the disclosure are set forth in thefollowing description and claims.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to”, andare not intended to (and do not) exclude other moieties, additives,components, integers or steps.

This patent application contains data indicating that the stability(resistance to deboronation) of organoboronic acids may be increased byproviding them in the form of salts with multivalent metals. The saltmay be an acid salt. This stabilisation technique forms part of thedisclosure and is applicable, inter alla, to organoboronic acidsdescribed under the heading “BACKGROUND” and to organoboronic acidsdescribed in publications mentioned under that heading.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC plot referred to in Example 13, showing an impurityprofile of encapsulated TRI 50c calcium salt after having beenmaintained in blister packaging for 1.5 month at 25° C. and 60% relativehumidity.

FIG. 2 is an HPLC plot referred to in Example 13, showing an impurityprofile of encapsulated TRI 50c calcium salt after having beenmaintained in blister packaging for 1.5 month at 40° C. and 75% relativehumidity.

FIG. 3 is an HPLC plot referred to in Example 13, showing an impurityprofile of encapsulated TRI 50c calcium salt after having beenmaintained absent blister packaging for 1.5 month at 40° C. and 75%relative humidity.

FIG. 4 is a chart referred to in Example 14, showing the results of athrombin amidolytic assay of TRI 1405 (TRI 50c magnesium salt) and TRI50b, where Vmax is the maximum rate of reaction measured by amidolyticassay.

FIG. 5 is a plot referred to in Example 25, showing oral phase clearanceand kinetics following p.o. dosing with TRI 50b or TRI 50c.

FIG. 6 is a second plot referred to in Example 25, showing oral phaseclearance and kinetics following intraduodenal dosing with TRI 50b orTRI 50c.

DETAILED DESCRIPTION OF SEVERAL EXAMPLES Glossary

The following terms and abbreviations are used in this specification:

The expression “acid salt” as applied to a salt of a boronic acid refersto salts of which a single —OH group of the trigonally-represented acidgroup —B(OH)₂ is deprotonated. Thus salts wherein the boronate groupcarries a single negative charge and may be represented as —B(OH)(O⁻) oras [—B(OH)₃]⁻ are acid salts. The expression encompasses salts of ametal having a valency n wherein the molar ratio of boronic acid tocation is approximately n to 1. In practical terms, the observedstoichiometry is unlikely to be exactly n:1 but will be consistent witha notional n:1 stoichiometry. For example, the observed mass of themetal might vary from the calculated mass for a n:1 stoichiometry by nomore than about 10%, e.g. no more than about 7.5%; in some cases anobserved mass of a metal might vary from the calculated mass by no morethan about 1%. Calculated masses are suitably based on the trigonal formof the boronate. (At an atomic level, a salt stoichiometricallyconsistent with being an acid salt might contain boronates in a mix ofprotonation states, whose average approximates to single deprotonationand such “mixed” salts are included in the term “acid salt”). Examplesof acid salts are hemimagnesium salts and hemicalcium salts.

α-Aminoboronic acid or Boro(aa) refers to an amino acid in which the CO₂group has been replaced by BO₂.

The term “amino-group protecting moiety” refers to any group used toderivatise an amino group, especially an N-terminal amino group of apeptide or amino acid. Such groups include, without limitation, alkyl,acyl, alkoxycarbonyl, aminocarbonyl, and sulfonyl moieties. However, theterm “amino-group protecting moiety” is not intended to be limited tothose particular protecting groups that are commonly employed in organicsynthesis, nor is it intended to be limited to groups that are readilycleavable.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expression “thrombin inhibitor” refers to a product which, withinthe scope of sound pharmacological judgement, is potentially or actuallypharmaceutically useful as an inhibitor of thrombin, and includesreference to substance which comprises a pharmaceutically active speciesand is described, promoted or authorised as a thrombin inhibitor. Suchthrombin inhibitors may be selective, that is they are regarded, withinthe scope of sound pharmacological judgement, as selective towardsthrombin in contrast to other proteases; the term “selective thrombininhibitor” includes reference to substance which comprises apharmaceutically active species and is described, promoted or authorisedas a selective thrombin inhibitor. The terms “protease inhibitor” and“selective protease inhibitor” have analogous meanings.

The term “heteroaryl” refers to a ring system which has at least one(e.g. 1, 2 or 3) in-ring heteroatoms and has a conjugated in-ring doublebond system. The term “heteroatom” includes oxygen, sulfur and nitrogen,of which sulfur is sometimes less preferred.

“Natural amino acid” means an L-amino acid (or residue thereof) selectedfrom the following group of neutral (hydrophobic or polar), positivelycharged and negatively charged amino acids:

-   -   Hydrophobic Amino Acids    -   A=Ala=alanine    -   V=Val=valine    -   I=Ile=isoleucine    -   L=Leu=leucine    -   M=Met=methionine    -   F=Phe=phenylalanine    -   P=Pro=proline    -   W=Trp=tryptophan    -   Polar (Neutral or Uncharged) Amino Acids    -   N=Asn=asparagine    -   C=Cys=cysteine    -   Q=Gln=glutamine    -   G=Gly=glycine    -   S=Ser=serine    -   T=Thr=threonine    -   Y=Tyr=tyrosine    -   Positively Charged (Basic) Amino Acids    -   R=Arg=arginine    -   H=His=histidine    -   K=Lys=lysine    -   Negatively Charged Amino Acids    -   D=Asp=aspartic acid    -   E=Glu=glutamic acid.

ACN=acetonitrile

Amino acid=α-amino acid

Cbz=benzyloxycarbonyl

Cha=cyclohexylalanine (a hydrophobic unnatural amino acid)

Charged (as applied to drugs or fragments of drug molecules, e.g. aminoacid residues)=carrying a charge at physiological pH, as in the case ofan amino, amidino or carboxy group

Dcha=dicyclohexylalanine (a hydrophobic unnatural amino acid)

Dpa=diphenylalanine (a hydrophobic unnatural amino acid)

Drug=a pharmaceutically useful substance, whether the active in vivoprinciple or a prodrug

i.v.=intravenous

Mpg=3-methoxypropylglycine (a hydrophobic unnatural amino acid)

Multivalent=valency of at least two, for example two or three

Neutral (as applied to drugs or fragments of drug molecules, e.g. aminoacid residues)=uncharged=not carrying a charge at physiological pH

Pinac=Pinacol=2,3-dimethyl-2,3-butanediol

Pinanediol=2,3-pinanediol=2,6,6-trimethylbicyclo[3.1.1] heptane-2,3-diol

Pip=pipecolinic acid

p.o.=per os=by way of the mouth (thus an oral formulation isadministered p.o.)

s.c.=subcutaneous

THF=tetrahydrofuran

Thr=thrombin

The Compounds

The products of the disclosure comprise a salt of a pharmaceuticallyacceptable multivalent (at least divalent) metal and an organoboronicacid drug (where the term “drug” embraces prodrugs). As previouslystated, the term “salt” refers to a product containing a multivalentmetal and an organoboronate species, for example a product having thecharacteristics of a product of a reaction between an organoboronic acidand a base comprising a multivalent metal (for example a +2 ion); inparticular, such characteristics comprise the identity of themultivalent metal and of the drug species.

One class of products comprises those salts which are acid salts. Asecond class of products comprises those salts which, whether or notacid, are salts of a boronic acid of formula III. A third class ofproducts comprises all the salts in contexts relating to their oraladministration, for example when present in oral formulations.

The acid may for example be any boronic acid drug mentioned under theheading “BACKGROUND” or in any document referred to under that heading,e.g. it may be TRI 50c or LDP-341. It may be a boronic acid described inWO 01/02424. In this paragraph, reference to a boronic acid described inthe prior art includes reference to the free acids of boronate estersdescribed in the prior art. It may be any other boronic acid drug.

In certain embodiments the organoboronic acid is hydrophobic.

Disclosed herein are embodiments in which the organoboronic acidcomprises an aminoboronic acid linked through a peptide linkage to anorganic moiety, which organic moiety may be hydrophobic. The organicmoiety can comprise an amino acid whose C-terminal carboxy group formspart of said peptide linkage. The disclosure therefore includes salts ofcompounds of formula (XIII) and a multivalent, e.g. divalent, metal:

In formula (XII), G is an organic-moiety, for example comprisingtogether with —CO— a residue of an optionally N-terminally substitutedamino acid or peptide (e.g. dipeptide), a suitable N-terminalsubstituent being for example an X group as described below. R is a sidechain of an amino acid (whether natural or unnatural). G and R may behydrophobic. R may be an R¹ group as described below.

One specific class of salts comprises those wherein the organoboronicacid comprises a boropeptide or boropeptidomimetic. For example, in asub-class of these salts the organoboronic acid is of the formula (I):

where:

R¹ is H or a non-charged side group;

R² is H or C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen orsulfur and optionally substituted by a substituent selected from halo,hydroxy and trifluoromethyl;

-   -   or R¹ and R² together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur;

R³ is the same as or different from R¹ provided that no more than one ofR¹ and R² is H;

R⁴ is H or a C₁-C₁₃ hydrocarbyl group optionally containing in-chainoxygen or sulfur and optionally substituted by a substituent selectedfrom halo, hydroxy and trifluoromethyl;

-   -   or R³ and R⁴ together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur; and

R⁵ is X-E- wherein E is nothing or a hydrophobic moiety selected fromthe group consisting of amino acids (natural or unnatural) and peptidesof two or more amino acids (natural or unnatural) of which more thanhalf are hydrophobic, in which peptides the nitrogen(s) of the peptidelinkage(s) may be substituted by a C₁-C₁₃ hydrocarbyl optionallycontaining in-chain oxygen or sulfur and optionally substituted by asubstituent selected from halo, hydroxy and trifluoromethyl (an exampleof such an N-substituent is 1C to 6C alkyl), and X is H or anamino-protecting group.

Said C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen or sulfurmay be selected from alkyl; alkyl substituted by cycloalkyl, aryl orheterocyclyl; cycloalkyl; aryl; and/or heterocyclyl. Heterocyclyl may beheteroaryl.

R¹ may be non polar. In some embodiments, R¹ contains up to 20 carbonatoms. R¹ may have affinity for the S1 subsite of a protease.

In one class of compounds, R¹ is a moiety other than hydrogen selectedfrom a group of formula A or B:

—(CO)_(a)—(CH₂)_(b)-D_(c)-(CH₂)_(d)-E   (A)

—(CO)_(a)—(CH₂)_(b)D_(c)-C_(e)(E¹)(E²)(E³)   (B)

wherein

a is 0 or 1;

e is 1;

b and d are independently 0 or an integer such that (b+d) is from 0 to 4or, as the case may be, (b+e) is from 1 to 4;

c is 0 or 1;

D is O or S;

E is H, C₁-C₆ alkyl, or a saturated or unsaturated cyclic group whichnormally contains up to 14 members and particularly is a 5-6 memberedring (e.g. phenyl) or an 8-14 membered fused ring system (e.g.naphthyl), which alkyl or cyclic group is optionally substituted by upto 3 groups (e.g. 1 group) independently selected from C₁-C₆trialkylsilyl, —CN, —R¹³, —R¹²OR¹³, —R¹²COR¹³, —R¹²CO₂R¹³ and—R¹²O₂CR¹³, wherein R¹² is —(CH₂)_(f)— and R¹³ is —(CH₂)_(g)H or by amoiety whose non-hydrogen atoms consist of carbon atoms and in-ringheteroatoms and number from 5 to 14 and which contains a ring system(e.g. an aryl group) and optionally an alkyl and/or alkylene group,wherein f and g are each independently from 0 to 10, g particularlybeing at least 1 (although —OH may also be mentioned as a substituent),provided that (f+g) does not exceed 10, more particularly does notexceed 6 and most particularly is 1, 2, 3 or 4, and provided that thereis only a single substituent if the substituent is a said moietycontaining a ring system, or E is C₁-C₆ trialkylsilyl; and E¹, E² and E³are each independently selected from —R¹⁵ and -J-R¹⁵, where J is a 5-6membered ring and R¹⁵ is selected from C₁-C₆ trialkylsilyl, —CN, —R¹³,—R¹²OR¹³, —R¹²COR¹³, —R¹²CO₂R¹³, —R¹²O₂CR¹³, and one or two halogens(e.g. in the latter case to form a -J-R¹⁵ moiety which isdichlorophenyl), where R¹² and R¹³ are, respectively, an R¹² moiety andan R¹³ moiety as defined above (in some acids where E¹, E² and E³contain an R¹³ group, g is 0 or 1);

in which moiety of Formula (A) or (B) any ring is carbocyclic oraromatic, or both, and any one or more hydrogen atoms bonded to a carbonatom is optionally replaced by halogen, especially F.

In certain examples, a is 0. If a is 1, c may be 0. In particularexamples, (a+b+c+d) and (a+b+c+e) are no more than 4 and are moreespecially 1, 2 or 3. (a+b+c+d) may be 0.

Exemplary groups for E, E¹, E² and E³ include aromatic rings such asphenyl, naphthyl, pyridyl, quinolinyl and furanyl, for example;non-aromatic unsaturated rings, for example cyclohexenyl; saturatedrings such as cyclohexyl, for example. E may be a fused ring systemcontaining both aromatic and non-aromatic rings, for example fluorenyl.One class of E, E¹, E² and E³ groups are aromatic (includingheteroaromatic) rings, especially 6-membered aromatic rings. In somecompounds, E¹ is H whilst E² and E³ are not H; in those compounds,examples of E² and E³ groups are phenyl (substituted or unsubstituted)and C₁-C₄ alkyl, e.g. methyl.

In one class of embodiments, E contains a substituent which is C₁-C₆alkyl, (C₁-C₅ alkyl)carbonyl, carboxy C₁-C₅ alkyl, aryl (includingheteroaryl), especially 5-membered or preferably 6-membered aryl (e.g.phenyl or pyridyl), or arylalkyl (e.g. arylmethyl or arylethyl wherearyl may be heterocyclic and is preferably 6-membered).

In another class of embodiments, E contains a substituent which is OR¹³,wherein R¹³ can be a 6-membered ring, which may be aromatic (e.g.phenyl) or is alkyl (e.g. methyl or ethyl) substituted by such a6-membered ring.

A class of moieties of formula A or B are those in which E is a6-membered aromatic ring optionally substituted, particularly at the2-position or 4-position, by —R¹³ or —OR¹³.

The disclosure also includes salts in which R¹ comprises a cyclic groupin which 1 or 2 hydrogens have been replaced by halogen, e.g. F or Cl.

The disclosure further includes a class of salts in which the R¹ groupsof formula (A) or (B) are of the following formulae (C), (D) or (E):

wherein q is from 0 to 5, e.g. is 0, 1 or 2, and each T is independentlyhydrogen, one or two halogens (e.g. F or Cl), —SiMe₃, —CN, —R¹³, —OR¹³,—COR¹³, —CO₂R¹³ or —O₂CR¹³. In some embodiments of structures (D) and(E), T is at the 4-position of the phenyl group(s) and is —R¹³, —OR¹³,—COR¹³, —CO₂R¹³ or —O₂CR¹³, and R¹³ is C₁-C₁₀ alkyl and moreparticularly C₁-C₆ alkyl. In one sub-class, T is —R¹³ or —OR¹³, forexample in which f and g are each independently 0, 1, 2 or 3; in some R¹groups of this sub-class, T is —R¹²OR¹³ and R¹³ is H.

In one class of the moieties, R¹ is of formula (C) and each T isindependently R¹³ or OR¹³ and R¹³ is C₁-C₄ alkyl. In some of thesecompounds, R¹³ is branched alkyl and in others it is straight chain. Insome moieties, the number of carbon atoms is from 1 to 4. In anotherclass of moieties, R¹ is of formula (E) and T is —CN or one or twohalogens; in these compounds, q may be 0 or 1, for example.

One class of compounds have R² as H and R³ as not H. Where R³ is not H,it is preferably conjoined with R⁴ to form a said moiety. Where R³ is H,R⁴ is preferably a said hydrocarbyl group, for example a C₄-C₆hydrocarbyl group comprising a C₅-C₆ hydrocarbyl ring; the hydrocarbylgroup may be saturated, for example an exemplary R⁴ group for thesecompounds is cyclopentyl.

In particular examples, R⁴ is H or R³ and R⁴ together form a said C₁-C₁₃moiety.

Where R³ does not join together with R⁴ to form a said C₁-C₁₃ moiety, insome embodiments it contains up to 20 carbon atoms.

R³ may be a group of formula (A) or (B) as defined above, for example agroup of formula (C), (D) or (E). In one class of compounds, R³ is offormula (C).

In one class of salts E is nothing.

In another class, E is comprises a sequence of one or more hydrophobicamino acids, for example such hydrophobic amino acids may have a sidechain having up to 20 carbon atoms. In some compounds, E comprises asequence of one or more hydrophobic amino acids (e.g. 1 amino acid) eachof which has a side chain of formula (A) or (B) as defined above, e.g. agroup of formula (C), (D) or (E), or is an imino acid, for example ofthe type formed when R¹ and R² of Formula (I) are joined together. Inone class of salts, E consists of an amino acid having a side chain offormula (D); In another class of salts, E consists of an amino acidhaving a side chain is of formula (E).

One specific class of salts comprises those in which the organoboronicacid is of the formula (II): wherein

R⁷ is X-E′- wherein X is hydrogen or an amino-protecting group and E′ isabsent or is a hydrophobic amino acid;

R⁸ is an optionally substituted moiety containing from 1 to 5 carbonatoms selected from the group consisting of alkyl, alkoxy andalkoxyalkyl, the optional substituents being hydroxy or, preferably,halogen (F, Cl, Br, I) and the alkyl moieties being branched or straightchain; and aa^(h) is a hydrophobic amino acid, or is glycineN-substituted by a C₁-C₁₃ hydrocarbyl group optionally containingin-chain oxygen or sulfur and optionally substituted by a substituentselected from halo, hydroxy and trifluoromethyl.

R⁷ may be X—, or X-Phe or X-Dpa.

R⁸ is preferably not substituted. R⁸ is preferably a C₄ group, e.g.alkyl or alkoxyalkyl, such as 2-methylpropyl or 3-methoxypropyl, forexample. In variants of Formula (II), R⁸ is phenyl or benzyl, in eithercase optionally substituted by —CN or by one or two halogens (e.g.chlorine).

When aa^(h) is N-substituted glycine, the N-substituent is for example aC₃-C₆ hydrocarbyl group comprising a C₃-C₆ hydrocarbyl ring; thehydrocarbyl group may be saturated, for example an exemplary R⁴ groupfor these compounds is cycloalkyl, e.g. cyclopentyl.

The hydrophobic amino acids may be the same or different and for examplebe selected from amino acids having a side chain of formula (A) or (B)as defined above, e.g. of formula (C), (D) or (E), and from imino acidsas described previously. The disclosure includes a class of saltswherein the organoboronic acid is of formula (II) and the hydrophobicamino acids, being the same or different, have a side chain containingup to 20 carbon atoms and often containing up to 13 carbon atoms or areimino acids. The hydrophobic amino acids may have a side chain asdescribed previously for hydrophobic amino acids contained in thefragment X-E of Formula (I). In a subset of salts containing formula(II) acids, the hydrophobic amino acid is hydrocarbyl or heteroaryl, orwhich includes both hydrocarbyl and heteroaryl residues. The hydrocarbylresidues optionally contain in-chain oxygen; they may be substituted by,for example, halogen (e.g. 1, 2 or 3 halogen atoms) or hydroxy (butusually not more than one hydroxy group). Alternatively, hydrophobicamino acids may be proline or another imino acid.

In certain variants, R⁷ contains a hydrophobic amino acid which is notPro or another imino acid. In such embodiments, the hydrophobic aminoacid of R⁷ suitably has a side chain of formula (A) or (B) describedpreviously [e.g. of formula (D) or (E)].

aa^(h) may for example be a natural hydrophobic amino acid, e.g. Pro orPhe.

In certain examples X is R⁶—(CH₂)_(p)—C(O)—, R⁶—(CH₂)_(p)—S(O)₂—,R⁶—(CH₂)_(p)—NH—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— wherein p is 0, 1, 2, 3,4, 5 or 6 (of which 0 and 1 are preferred) and R⁶ is H or a 5 to13-membered cyclic group optionally substituted by 1, 2 or 3substituents selected from halogen, amino, nitro, hydroxy, a C₅-C₆cyclic group, C₁-C₄ alkyl and C₁-C₄ alkyl containing, and/or linked tothe 5 to 13-membered cyclic group through, an in-chain O, the aforesaidalkyl groups optionally being substituted by a substituent selected fromhalogen, amino, nitro, hydroxy and a C₅-C₆ cyclic group. Moreparticularly X is R⁶—(CH₂)_(p)—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— and p is 0or 1. Said 5 to 13-membered cyclic group is often aromatic orheteroaromatic, for example is a 6-membered aromatic or heteroaromaticgroup. In many cases, the group is not substituted.

Exemplary X groups are (2-pyrazine) carbonyl, (2-pyrazine) sulfonyl andbenzyloxycarbonyl.

The organoboronic acid may be a protease inhibitor, for example a serineprotease inhibitor. Thus the disclosure includes salts of a multivalentmetal and an organoboronic acid inhibitor of a coagulation serineprotease, for example thrombin or Factor Xa. As examples of suchorganoboronic acids may be mentioned peptide boronates, particularlydipeptides and tripeptides, which in either case may have a protectinggroup (a non-hydrogen X group) on the N-terminal amino moiety.

In a preferred class of boronic acids, which are anti-thrombotic andinclude TRI 50c, the acid has a neutral moiety capable of binding to thethrombin S1 subsite linked to a hydrophobic moiety capable of binding tothe thrombin S2 and S3 subsites. The acid may for example be of formula(III):

wherein

Y comprises a moiety which, together with the fragment —CH(R⁹)—B(OH)₂,has affinity for the substrate binding site of thrombin; and

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages (e.g. 1 or 2) and in which the total number of oxygen andcarbon atoms is 3, 4, 5 or 6 (e.g. 5) or R⁹ is —(CH₂)_(m)—W where m is2, 3, 4 or 5 (e.g. 4) and W is —OH or halogen (F, Cl, Br or I). Asexamples of straight chain alkyl interrupted by one or more etherlinkages (—O—) may be mentioned alkoxyalkyl (one interruption) andalkoxyalkoxyalkyl (two interruptions). R⁹ is an alkoxyalkyl group in onesubset of compounds, e.g. alkoxyalkyl containing 4 carbon atoms.

In a class of boronic acids, Y is linked to —CH(R⁹)—B(OH)₂ by a peptidelinkage. Such acids may be represented by formula (XII):

wherein

Y¹ comprises a hydrophobic moiety which, together with the aminoboronicacid residue —NHCH(R⁹)—B(OH)₂, has affinity for the substrate bindingsite of thrombin and R⁹ is as defined above.

Typically, therefore, the moiety represented by symbol Y in formula(III) comprises an amino acid residue (whether natural or unnatural)which binds to the S2 subsite of thrombin and is linked to the fragment—CH(R⁹)—B(OH)₂ by a peptide linkage, the amino acid residue beingN-terminally linked to a moiety which binds the 53 subsite of thrombin.

In one class of Formula (III) acids, Y is an optionally N-terminallyprotected dipeptide residue which binds to the 53 and 52 binding sitesof thrombin and is linked to —CH(R⁹)—B(OH)₂ by a peptide linkage, thepeptide linkages in the acid optionally and independently beingN-substituted by a C₁-C₁₃ hydrocarbyl optionally containing in-chainoxygen or sulfur and optionally substituted by a substituent selectedfrom halo, hydroxy and trifluoromethyl. The N-terminal protecting group,when present, may be a group X as defined above (other than hydrogen).Normally, the acid contains no N-substituted peptide linkages; wherethere is an N-substituted peptide linkage, the substituent is often 1Cto 6C alkyl. One class of acids has an N-terminal protecting group (e.g.an X group) and unsubstituted peptide linkages.

Where Y is a dipeptide residue, the S3-binding amino acid residue may beof (R)-configuration and/or the S2-binding residue may of(S)-configuration. The fragment —NHCH(R⁹)—B(OH) may of(R)-configuration. The disclosure is not restricted to chiral centres ofthese conformations, however.

The disclosure therefore includes medicaments comprising salts, e.g.metal salts, of organoboronic acids which are thrombin inhibitors,particularly selective thrombin inhibitors, having a neutral P1(S1-binding) moiety. For more information about moieties which bind tothe S3, S2 and S1 sites of thrombin, see for example Tapparelli C et al,Trends Pharmacol. Sci. 14: 366-376, 1993; Sanderson P et al, CurrentMedicinal Chemistry, 5: 289-304, 1998; Rewinkel J et al, CurrentPharmaceutical Design, 5:1043-1075, 1999; and Coburn C Exp. Opin. Ther.Patents 11(5): 721-738, 2001. The thrombin inhibitory salts of thedisclosure are not limited to those having S3, S2 and S1 affinity groupsdescribed in the publications listed in the preceding sentence.

The organoboronic acids which are thrombin inhibitors, for example theacids of formula (III), may have a Ki for thrombin of about 100 nM orless, e.g. about 20 nM or less.

A subset of the Formula (III) acids comprises the acids of Formula (IV):

X is a moiety bonded to the N-terminal amino group and may be H to formNH₂. The identity of X is not critical but may be a particular X moietydescribed above. In one example there may be mentionedbenzyloxycarbonyl.

aa¹ is an amino acid residue having a hydrocarbyl side chain containingno more than 20 carbon atoms (e.g. up to 15 and optionally up to 13 Catoms) and comprising at least one cyclic group having up to 13 carbonatoms. In certain examples, the cyclic group(s) of aa¹ have/has 5 or 6ring members. For instance, the cyclic group(s) of aa¹ may be arylgroups, particularly phenyl. Typically, there are one or two cyclicgroups in the aa¹ side chain. Certain side chains comprise, or consistof, methyl substituted by one or two 5- or 6-membered rings.

More particularly, aa¹ is Phe, Dpa or a wholly or partially hydrogenatedanalogue thereof. The wholly hydrogenated analogues are Cha and Dcha.

aa² is an imino acid residue having from 4 to 6 ring members.Alternatively, aa² is Gly N-substituted by a C₃-C₁₃ hydrocarbyl group,e.g. a C₃-C₈ hydrocarbyl group comprising a C₃-C₆ hydrocarbyl ring; thehydrocarbyl group may be saturated, for example exemplary N-substituentsare cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. As ahydrocarbyl group containing one or more unsaturated bonds may bementioned phenyl and methyl or ethyl substituted by phenyl, e.g.2-phenylethyl, as well as β,β-dialkylphenylethyl.

An exemplary class of products comprises those in which aa² is a residueof an imino acid of formula (V)

where R¹¹ is —CH₂—, CH₂—CH₂—, —S—CH₂— or —CH₂—CH₂—CH₂—, which group whenthe ring is 5 or 6-membered is optionally substituted at one or more—CH₂— groups by from 1 to 3 C₁-C₃ alkyl groups, for example to form theR¹¹ group —S—C(CH₃)₂—. Of these imino acids, azetidine-2-carboxylicacid, especially (s)-azetidine-2-carboxylic acid, and more particularlyproline are illustrative.

It will be appreciated from the above that a very preferred class ofproducts consists of those in which aa¹-aa² is Phe-Pro. In anotherpreferred class, aa¹-aa² is Dpa-Pro. In other products, aa¹-aa² isCha-Pro or Dcha-Pro. Of course, also included are corresponding productclasses in which Pro is replaced by (s)-azetidine-2-carboxylic acid.

R⁹ is as defined in relation to formula (III). In one class of compounds[whether of formula (III) or formula (IV)], R⁹ is a group of the formula—(CH₂)_(s)-Z. Integer s is 2, 3 or 4 and Z is —OH, —OMe, —OEt or halogen(F, Cl, I or, preferably, Br). Particularly illustrative Z groups are—OMe and —OEt, especially —OMe. In certain examples s is 3 for all Zgroups and, indeed, for all formula (III) or (IV) compounds. ParticularR⁹ groups are 2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 4-bromobutyl,4-chlorobutyl, 4-methoxybutyl and, especially, 3-bromopropyl,3-chloropropyl and 3-methoxypropyl. In a specific example, R⁹ is3-methoxypropyl. In another example, 2-Ethoxyethyl is the preferred R⁹group.

Accordingly, a specific class of salts consists of those of acids of theformula X-Phe-Pro-Mpg-B(OH)₂, especially Cbz-Phe-Pro-Mpg-B(OH)₂; alsoincluded are analogues of these compounds in which Mpg is replaced by aresidue with another of the R⁹ groups and/or Phe is replaced by Dpa oranother aa¹ residue.

The aa¹ moiety of the salt is preferably of (R)-configuration. The aa²moiety is preferably of (S)-configuration. Particularly preferred saltshave aa¹ of (R)-configuration and aa² of (S)-configuration. The chiralcentre —NH—CH(R¹)—B— is preferably of (R)-configuration. It isconsidered that commercial formulations will have the chiral centres in(R,S,R) arrangement, as for example in the case of salts ofCbz-Phe-Pro-BoroMpg-OH:

The disclosure includes multivalent metal salts ofCbz-(R)-Phe-(S)-Pro-(R)-boroMpg-OH (and of other compounds of theformula X—(R)-Phe-(S)-Pro-(R)-boroMpg-OH) which are at least 90% pure,e.g. at least 95% pure.

In broad terms, the salts described herein may be considered tocorrespond to reaction products of an organoboronic acid as describedabove with a base of a multivalent metal, i.e. a metal having a valencyof two or more; the salts are however not limited to products resultingfrom such a reaction and may be obtained by alternative routes. Themetal is especially:

1. a Group II metal (alkaline earth metal);

2. another pharmaceutically acceptable divalent metal, e.g. zinc; or

3. a Group III metal.

One exemplary class of salts comprises divalent metal salts. Aparticularly illustrative class of salts comprises the calcium salts.Another particularly illustrative class of salts comprises the magnesiumsalts. A further class of salts comprises the zinc salts.

Specific salts are of the acid boronate though in practice the acidsalts may contain a very small proportion of the doubly deprotonatedboronate. The term “acid boronate” refers to trigonal —B(OH)₂ groups inwhich one of the B—OH groups is deprotonated as well as to correspondingtetrahedral groups in equilibrium therewith. Acid boronates have astoichiometry consistent with single deprotonation.

The disclosure further includes therefore products (compositions ofmatter) which comprise salts which may be represented by formula (VI):

where M^(n+) is a divalent or trivalent metal cation, aa^(2′) is aresidue of an imino acid of formula V, n is 2 or 3 as the case may be,and aa¹, X and R⁹ are as defined above. As previously indicated, theboronate may comprise a tetrahedral species. Accordingly, illustrativeproducts have a stoichiometry consistent with the above formula.

The disclosure additionally includes calcium and magnesium salts ofboronic acid drugs having an observed stoichiometry consistent with thesalt being of (being representable by) the formula “(boronate⁻)₂ Ca²⁺”,or “(boronate⁻)₂ Mg²⁺”. In other salts of this type, the metal is zinc.One class of salts having such stoichiometry comprises salts of boronicacids of formula (IV), as for example in the case of a salt of theformula:

[Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)(O⁻)]₂ Ca²⁺,

such salt being designated TGN 167. The disclosure includes salts of theabove formula in which Ca²⁺ is replaced by Mg²⁺. Also included arecorresponding zinc salts. It will be understood that the aboverepresentation is a notional representation of a product whose observedstoichiometry is unlikely to be literally and exactly 2:1. In the aboveformula, the trigonally-represented boronate represents boronates whichare trigonal, tetrahedral or mixed trigonal/tetrahedral.

Particularly exemplary are products which comprise:

(i) species selected from (a) acids of formula (IX):X—(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂ where X is H or an amino-protectinggroup, especially Cbz, (b) boronate anions thereof, and (c) anyequilibrium form of the aforegoing (e.g. an anhydride); and

(ii) divalent metal ions, particularly calcium ions, in combination withsaid species, the species and the metal ions having an observedstoichiometry consistent with a notional species:metal stoichiometry of2:1.

Considering the metals in turn:

1. Divalent, e.g. Alkaline Earth Metal (Group II Metal) Salts

One example of a divalent metal is calcium. Another suitable divalentmetal is magnesium. Also contemplated is zinc. The divalent metals areusually used in a boronic acid:metal ratio of substantially 2:1, inorder to achieve the preferred monovalent boronate moiety. Saltscontaining mixtures of divalent metals, e.g. mixtures of alkaline earthmetals, are also contemplated.

Further disclosed are products (compositions of matter) which comprisesalts which may be represented by the formula (VII):

where M²⁺ is a divalent metal cation, e.g. an alkaline earth metal orzinc cation, and aa¹, aa^(2′), X and R⁹ are as defined above, as well assalts in which both hydroxy groups of the boronate group aredeprotonated and mixtures of such salts. As previously indicated, theboronate may comprise a tetrahedral species.

2. Group III Metals

Suitable Group III metals include aluminium and gallium. Saltscontaining mixtures of Group III metals are also contemplated.

The disclosure includes products comprising salts of the formula (VIII):

where M³⁺ is a Group III metal ion and aa¹, aa^(2′), X and R⁹ are asdefined above, as well as salts in which both hydroxy groups of theboronate group are in salt form and mixtures of such salts. Aspreviously indicated, the boronate may comprise a tetrahedral species.

The salts in solid form may contain a solvent, e.g. water. There areincluded a class of products in which the salts are essentiallyanhydrous. Also included is a class in which the salts are hydrates.

Use of the Products of the Disclosure

The salts are useful for formulations, especially for oral formulations,for administering the drug part of the salt. Typically, they are usefulas protease inhibitors.

The Salts of Thrombin Inhibitors

As described above, disclosed herein are salts of boronic acids whichare thrombin inhibitors. For example, the salts of the boronic acids offormula (IV) are potent thrombin inhibitors. They are therefore usefulfor inhibiting thrombin. The disclosure therefore provides thrombininhibitory compounds which have potential for controlling haemostasisand especially for inhibiting coagulation, for example in the treatmentor prevention of secondary events after myocardial infarction. Themedical use of the compounds may be prophylactic (including to treatthombosis as well as to prevent occurrence of thrombosis) as well astherapeutic (including to prevent re-occurrence of thrombosis orsecondary thrombotic events).

The thrombin inhibitory salts may be employed when an anti-thrombogenicagent is needed. Further, it has been found that these salts arebeneficial in that the class is useful for treating arterial thrombosisby therapy or prophylaxis. The disclosed thrombin inhibiting salts arethus indicated in the treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues of animals including man. Theterm “thrombosis” includes inter alia atrophic thrombosis, arterialthrombosis, cardiac thrombosis, coronary thrombosis, creepingthrombosis, infective thrombosis, mesenteric thrombosis, placentalthrombosis, propagating thrombosis, traumatic thrombosis and venousthrombosis.

It is known that hypercoagulability may lead to thromboembolic diseases.

Examples of venous thromboembolism which may be treated or preventedwith compounds of the disclosure include obstruction of a vein,obstruction of a lung artery (pulmonary embolism), deep vein thrombosis,thrombosis associated with cancer and cancer chemotherapy, thrombosisinherited with thrombophilic diseases such as Protein C deficiency,Protein S deficiency, antithrombin III deficiency, and Factor V Leiden,and thrombosis resulting from acquired thrombophilic disorders such assystemic lupus erythematosus (inflammatory connective tissue disease).Also with regard to venous thromboembolism, compounds of the disclosureare useful for maintaining patency of indwelling catheters.

Examples of cardiogenic thromboembolism which may be treated orprevented with compounds of the disclosure include thromboembolic stroke(detached thrombus causing neurological affliction related to impairedcerebral blood supply), cardiogenic thromboembolism associated withatrial fibrillation (rapid, irregular twitching of upper heart chambermuscular fibrils), cardiogenic thromboembolism associated withprosthetic heart valves such as mechanical heart valves, and cardiogenicthromboembolism associated with heart disease.

Examples of conditions involving arterial thrombosis include unstableangina (severe constrictive pain in chest of coronary origin),myocardial infarction (heart muscle cell death resulting frominsufficient blood supply), ischemic heart disease (local ischemia dueto obstruction (such as by arterial narrowing) of blood supply),reocclusion during or after percutaneous transluminal coronaryangioplasty, restenosis after percutaneous transluminal coronaryangioplasty, occlusion of coronary artery bypass grafts, and occlusivecerebrovascular disease. Also with regard to arterio-venous (mixed)thrombosis, anti-thrombotic compounds of the disclosure are useful formaintaining patency in arteriovenous shunts.

Other conditions associated with hypercoagulability and thromboembolicdiseases which may be mentioned inherited or acquired deficiencies inheparin cofactor II, circulating antiphospholipid antibodies (Lupusanticoagulant), homocysteinemia, heparin induced thrombocytopenia anddefects in fibrinolysis.

Particular uses which may be mentioned include the therapeutic and/orprophylactic treatment of venous thrombosis and pulmonary embolism.Preferred indications envisaged for the anti-thrombotic products of thedisclosure (notably the salts of the boronic acids of formula IV, forexample the salts of TRI 50c) include:

-   -   Prevention of venous thromboembolic events (e.g. deep vein        thrombosis and/or pulmonary embolism). Examples include patients        undergoing orthopaedic surgery such as total hip replacement,        total knee replacement, major hip or knee surgery; patients        undergoing general surgery at high risk for thrombosis, such as        abdominal or pelvic surgery for cancer; and in patients        bedridden for more than 3 days and with acute cardiac failure,        acute respiratory failure, infection.    -   Prevention of thrombosis in the haemodialysis circuit in        patients, in patients with end stage renal disease.    -   Prevention of cardiovascular events (death, myocardial        infarction, etc.) in patients with end stage renal disease,        whether or not requiring haemodialysis sessions.    -   Prevention of venous thrombo-embolic events in patients        receiving chemotherapy through an indwelling catheter.    -   Prevention of thromboembolic events in patients undergoing lower        limb arterial reconstructive procedures (bypass,        endarteriectomy, transluminal angioplasty, etc.).    -   Treatment of venous thromboembolic events.    -   Prevention of cardiovascular events in acute coronary syndromes        (e.g. unstable angina, non Q wave myocardial        ischaemia/infarction), in combination with another        cardiovascular agent, for example aspirin (acetylsalicylic acid;        aspirin is a registered trade mark in Germany), thrombolytics        (see below for examples), antiplatelet agents (see below for        examples).    -   Treatment of patients with acute myocardial infarction in        combination with acetylsalicylic acid, thrombolytics (see below        for examples).

The presently disclosed thrombin inhibitors are thus indicated both inthe therapeutic and/or prophylactic treatment of all the aforesaiddisorders.

In one method, the presently disclosed thrombin inhibitors are used forthe treatment of patients by haemodialysis, by providing the product inthe dialysis solution, as described in relation to other thrombininhibitors in WO 00/41715. The present disclosure therefore includesdialysing solutions and dialysing concentrates which comprise thepresently disclosed anti-thrombotic product, as well as a method oftreatment by dialysis of a patient in need of such treatment, whichmethod comprises the use of a dialysing solution including a lowmolecular weight thrombin inhibitor. Also included is the use of ananti-thrombotic product of the disclosure for the manufacture of amedicament for the treatment by dialysis of a patient, in which theanti-thrombotic product of the disclosure is provided in the dialysingsolution.

In another method, the presently disclosed thrombin inhibitors are usedto combat undesirable cell proliferation, as described in relation toother thrombin inhibitors in WO 01/41796. The undesirable cellproliferation is typically undesirable hyperplastic cell proliferation,for example proliferation of smooth muscle cells, especially vascularsmooth muscle cells. The thrombin inhibitors particularly findapplication in the treatment of intimal hyperplasia, one component ofwhich is proliferation of smooth muscle cells. Restenosis can beconsidered to be due to neointimal hyperplasia; accordingly intimalhyperplasia in the present context includes restenosis.

The thrombin inhibitors are also contemplated for the treatment ofischemic disorders. More particularly, they may be used in the treatment(whether therapeutic or prophylactic) of an ischemic disorder in apatient having, or at risk of, non-valvular atrial fibrillation (NVAF)as described in relation to other thrombin inhibitors in WO 02/36157.Ischemic disorders are conditions whose results include a restriction inblood flow to a part of the body. The term will be understood to includethrombosis and hypercoagulability in blood, tissues and/or organs.Particular uses that may be mentioned include the prevention and/ortreatment of ischemic heart disease, myocardial infarction, systemicembolic events in e.g. the kidneys or spleen, and more particularly ofcerebral ischemia, including cerebral thrombosis, cerebral embolismand/or cerebral ischemia associated with non-cerebral thrombosis orembolism (in other words the treatment (whether therapeutic orprophylactic) of thrombotic or ischemic stroke and of transient ischemicattack), particularly in patients with, or at risk of, NVAF.

The thrombin inhibitors are also contemplated for the treatment ofrheumatic/arthritic disorders, as described in relation to otherthrombin inhibitors in WO 03/007984. Thus, a thrombin inhibitory saltmay be used in the treatment of chronic arthritis, rheumatoid arthritis,osteoarthritis or ankylosing spondylitis

Moreover, the thrombin inhibitors of the disclosure are expected to haveutility in prophylaxis of re-occlusion (i.e. thrombosis) afterthrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronarybypass operations; the prevention of re-thrombosis after microsurgeryand vascular surgery in general. Further indications include thetherapeutic and/or prophylactic treatment of disseminated intravascularcoagulation caused by bacteria, multiple trauma, intoxication or anyother mechanism; anticoagulant treatment when blood is in contact withforeign surfaces in the body such as vascular grafts, vascular stents,vascular catheters, mechanical and biological prosthetic valves or anyother medical device; and anticoagulant treatment when blood is incontact with medical devices outside the body such as duringcardiovascular surgery using a heart-lung machine or in haemodialysis.

The thrombin inhibitors are further indicated in the treatment ofconditions where there is an undesirable excess of thrombin withoutsigns of hypercoagulability, for example in neurodegenerative diseasessuch as Alzheimer's disease. In addition to its effects on thecoagulation process, thrombin is known to activate a large number ofcells (such as neutrophils, fibroblasts, endothelial cells and smoothmuscle cells). Therefore, the presently disclosed compounds may also beuseful for the therapeutic and/or prophylactic treatment of idiopathicand adult respiratory distress syndrome, pulmonary fibrosis followingtreatment with radiation or chemotherapy, septic shock, septicaemia,inflammatory responses, which include, but are not limited to, edema,acute or chronic atherosclerosis such as coronary arterial disease,cerebral arterial disease, peripheral arterial disease, reperfusiondamage, and restenosis after percutaneous trans-luminal angioplasty(PTA).

The thrombin inhibitory salts may also be useful in the treatment ofpancreatitis.

The salts of the boronic acids of formula (III) are further consideredto be useful for inhibiting platelet procoagulant activity. Alsoprovided is a method for inhibiting platelet pro-coagulant activity byadministering a salt of a formula (III) or formula (IV) boronic acid toa mammal at risk of, or suffering from, arterial thrombosis,particularly a human patient. Further provided is the use of such saltsfor the manufacture of medicaments for inhibiting platelet procoagulantactivity.

The use of the formula (III) or formula (IV) products as inhibitors ofplatelet pro-coagulant activity is predicated on the observation thatthe formula (III) or formula (IV) acids are effective at inhibitingarterial thrombosis as well as venous thrombosis.

Indications involving arterial thrombosis include acute coronarysyndromes (especially myocardial infarction and unstable angina),cerebrovascular thrombosis and peripheral arterial occlusion andarterial thrombosis occurring as a result of atrial fibrillation,valvular heart disease, arterio-venous shunts, indwelling catheters orcoronary stents. Accordingly, in another aspect, there is provided amethod of treating a disease or condition selected from this group ofindications, comprising administering to a mammal, especially a humanpatient, a thrombin inhibitory salt according to the present disclosure.The disclosure includes products for use in an arterial environment,e.g. a coronary stent or other arterial implant, having a coating whichcomprises an antithrombin salt according to the present disclosure.

The salts of the formula (III) or formula (IV) boronic acids may be usedprophylactically to treat an individual believed to be at risk ofsuffering from arterial thrombosis or a condition or disease involvingarterial thrombosis or therapeutically (including to preventre-occurrence of thrombosis or secondary thrombotic events).

The disclosure therefore includes the use of selective thrombininhibitors (organoboronic acid salts) described herein for treatment ofthe above disorders by prophylaxis or therapy as well as their use inpharmaceutical formulations and the manufacture of pharmaceuticalformulations.

In the case of those uses described above which are anti-coagulant innature, there may be used in place of a thrombin inhibitor of thedisclosure another anti-coagulant salt of the disclosure.

Administration and Pharmaceutical Formulations

The salts may be administered to a host, for example, in the case wherethe drug has anti-thrombogenic activity, to obtain an anti-thrombogeniceffect. In the case of larger animals, such as humans, the salts may beadministered alone or in combination with pharmaceutically acceptablediluents, excipients or carriers. The term “pharmaceutically acceptable”includes acceptability for both human and veterinary purposes, of whichacceptability for human pharmaceutical use is preferred. In the case oforal administration, the compounds, particularly the salts of amino- orpeptido-boronic acids, are preferably administered in a form whichprevents the salt from contact with the acidic gastric juice, such asenterically coated formulations, which thus prevent release of the saltuntil it reaches the duodenum.

The enteric coating is suitably made of carbohydrate polymers orpolyvinyl polymers, for example. Examples of enteric coating materialsinclude, but are not limited to, cellulose acetate phthalate, celluloseacetate succinate, cellulose hydrogen phthalate, cellulose acetatetrimellitate, ethyl cellulose, hydroxypropyl-methylcellulose phthalate,hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, starch acetate phthalate, amylose acetate phthalate,polyvinyl acetate phthalate, polyvinyl butyrate phthalate,styrene-maleic acid copolymer, methyl-acrylate-methacrylic acidcopolymer (MPM-05), methylacrylate-methacrylic acid-methylmethacrylatecopolymer (MPM-06), and methylmethacrylate-methacrylic acid co-polymer(Eudragit® L & S). Optionally, the enteric coating contains aplasticiser. Examples of the plasticiser include, but are not limitedto, triethyl citrate, triacetin, and diethyl phthalate.

The presently disclosed anti-thrombotic salts may be combined and/orco-administered with any cardiovascular treatment agent. There are largenumbers of cardiovascular treatment agents available in commercial use,in clinical evaluation and in pre-clinical development, which could beselected for use with the presently disclosed product for the preventionof cardiovascular disorders by combination drug therapy. Such agent canbe one or more agents selected from, but not limited to several majorcategories, namely, a lipid-lowering drug, including an IBAT (ilealNa⁺/bile acid cotransporter) inhibitor, a fibrate, niacin, a statin, aCETP (cholesteryl ester transfer protein) inhibitor, and a bile acidsequestrant, an anti-oxidant, including vitamin E and probucol, aIIb/IIIa antagonist (e.g. abciximab, eptifibatide, tirofiban), analdosterone inhibitor (e.g. spirolactone and epoxymexrenone), anadenosine A2 receptor antagonist (e.g. losartan), an adenosine A3receptor agonist, a beta-blocker, acetylsalicylic acid, a loop diureticand an ACE (angiotensin converting enzyme) inhibitor.

The anti-thrombotic salts may be combined and/or co-administered withany antithrombotic agent with a different mechanism of action, such asthe antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel,thromboxane receptor and/or synthetase inhibitors, prostacyclin mimeticsand phosphodiesterase inhibitors and ADP-receptor (P₂ T) antagonists.

The thrombin inhibitory salts may further be combined and/orco-administered with thrombolytics such as tissue plasminogen activator(natural, recombinant or modified), streptokinase, urokinase,prourokinase, anisoylated plasminogen-streptokinase activator complex(APSAC), animal salivary gland plasminogen activators, and the like, inthe treatment of thrombotic diseases, in particular myocardialinfarction.

The anti-thrombotic salts may be combined and/or co-administered with acardioprotectant, for example an adenosine A1 or A3 receptor agonist.

There is also provided a method for treating an inflammatory disease ina patient that comprises treating the patient with an anti-thromboticproduct and an NSAID, e.g., a COX-2 inhibitor. Such diseases include butare not limited to nephritis, systemic lupus, erythematosus, rheumatoidarthritis, glomerulonephritis, vasculitis and sacoidosis. Accordingly,the anti-thrombotic salts of the disclosure may be combined and/orco-administered with an NSAID.

Typically, therefore, the salts of the formula (III) and formula (IV)acids may be administered to a host to obtain a thrombin-inhibitoryeffect, or in any other thrombin-inhibitory or anti-thrombotic contextmentioned herein.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activecompound(s) that is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration (referred to herein as a “therapeutically effectiveamount”). The selected dosage level will depend upon the activity of theparticular compound, the severity of the condition being treated and thecondition and prior medical history of the patient being treated.However, it is within the skill of the art to start doses of thecompound at levels lower than required for to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

For example, it is currently contemplated that, in the case of oraladministration of salts of TRI 50c, the salts might for instance beadministered in an amount of from 0.5 to 2.5 mg/Kg twice daily,calculated as TRI 50c. Other salts might be administered in equivalentmolar amounts. However, the presently described methods are not limitedto administration in such quantities or regimens and includes dosagesand regimens outside those described in the previous sentence.

According to a further aspect there is provided an oral pharmaceuticalformulation including a product as described herein, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier.

Solid dosage forms for oral administration include capsules, tablets(also called pills), powders and granules. In such solid dosage forms,the active compound is typically mixed with at least one inert,pharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or one or more: a) fillers or extenders suchas starches, lactose, sucrose, glucose, mannitol and silicic acid; b)binders such as carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose and acacia; c) humectants such asglycerol; d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates and sodiumcarbonate; e) solution retarding agents such as paraffin; f) absorptionaccelerators such as quaternary ammonium compounds; g) wetting agentssuch as cetyl alcohol and glycerol monostearate; h) absorbents such askaolin and bentonite clay and i) lubricants such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate and mixtures thereof. In the case of capsules and tablets, thedosage form may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polyethylene glycol, for example.

Suitably, the oral formulations may contain a dissolution aid. Thedissolution aid is not limited as to its identity so long as it ispharmaceutically acceptable. Examples include nonionic surface activeagents, such as sucrose fatty acid esters, glycerol fatty acid esters,sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethyleneglycol, polyoxyethylene hydrogenated castor oil, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene alkyl ethers,methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers,polyethylene glycol fatty acid esters, polyoxyethylene alkylamines,polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylenecopolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritolfatty acid esters, propylene glycol monofatty acid esters,polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylenesorbitol fatty acid esters, fatty acid alkylolamides, and alkylamineoxides; bile acid and salts thereof (e.g., chenodeoxycholic acid, cholicacid, deoxycholic acid, dehydrocholic acid and salts thereof, andglycine or taurine conjugate thereof); ionic surface active agents, suchas sodium laurylsulfate, fatty acid soaps, alkylsulfonates,alkylphosphates, ether phosphates, fatty acid salts of basic aminoacids; triethanolamine soap, and alkyl quaternary ammonium salts; andamphoteric surface active agents, such as betaines and aminocarboxylicacid salts.

The active compounds may also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide,oils (in particular, cottonseed, groundnut, corn, germ, olive, castor,and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan and mixtures thereof. Besidesinert diluents, the oral compositions may also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavouring and perfuming agents. Suspensions, in addition to the activecompounds, may contain suspending agents such as ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanthand mixtures thereof.

The presently disclosed product may be presented as solids in finelydivided solid form, for example they may be micronised. Powders orfinely divided solids may be encapsulated.

The active compound may be given as a single dose, in multiple doses oras a sustained release formulation.

It will be understood from the aforegoing that there are providedpharmaceutical products comprising an alkaline earth metal salt,particularly calcium salt, of a boronic acid of Formula (III) or (IV) indry fine particle form, suitable for oral administration. The alkalineearth metal salt is suitably an acid salt.

Synthesis

1. Peptide/Peptidomimetic Synthesis

The synthesis of boropeptides, including, for example,Cbz-D-Phe-Pro-BoroMpg-OPinacol is familiar to those skilled in the artand described in the prior art mentioned above, including Claeson et al(U.S. Pat. No. 5,574,014 and others) and Kakkar et al (WO 92/07869 andfamily members including U.S. Pat. No. 5,648,338). It is described alsoby Elgendy et al Adv. Exp. Med. Biol. (USA) 340:173-178, 1993; Claeson,G. et al Biochem. J. 290:309-312, 1993; Deadman et al J. EnzymeInhibition 9:29-41, 1995, and by Deadman et al J. Med. Chem.38:1511-1522, 1995.

Stereoselective synthesis with S or R configuration at the chiralB-terminal carbon may be conducted using established methodology(Elgendy et al Tetrahedron. Lett. 33:4209-4212, 1992; WO 92/07869 andfamily members including U.S. Pat. No. 5,648,338) using (+) or(−)-pinanediol as the chiral director (Matteson et al J. Am. Chem. Soc.108:810-819, 1986; Matteson et al Organometallics. 3:1284-1288, 1984).Another approach is to resolve the requisite aminoboronate intermediate(e.g. Mpg-BOPinacol) to selectively obtain the desired (R)-isomer andcouple it to the dipeptide moiety (e.g. Cbz-(R)-Phe-(S)-Pro, which isthe same as Cbz-D-Phe-L-Pro) which will form the remainder of themolecule.

The reader is referred also to other prior documents mentionedpreviously in this specification, for example the US patents of Adams etal.

The boropeptides may be synthesised initially in the form of boronicacid esters, particularly esters with diols. Such diol esters may beconverted to the peptide boronic acid as described next.

2. Ester to Acid Conversion

A peptide boronate ester such as Cbz-(R)-Phe-Pro-BoroMpg-OPinacol may behydrolysed to form the corresponding acid.

A novel technique for converting a diol ester of a peptide boronic acidof formula (I) into the acid comprises dissolving the diol ester in anether and particularly a dialkyl ether, reacting the thus-dissolved diolwith a diolamine, for example a dialkanolamine, to form a productprecipitate, recovering the precipitate, dissolving it in a polarorganic solvent and reacting the thus-dissolved product with an aqueousmedium, e.g. an aqueous acid, to form the peptide boronic acid. Theboronic acid may be recovered from the organic layer of the mixtureresulting from the reaction, for example by removing the solvent, e.g.by evaporation under vacuum or distillation. The reaction between thediol ester and the diolamine may be carried out under reflux, forexample.

The identity of the diol is not critical. As suitable diols may bementioned aliphatic and aromatic compounds having hydroxy groups thatare substituted on adjacent carbon atoms or on carbon atoms substitutedby another carbon. That is to say, suitable diols include compoundshaving at least two hydroxy groups separated by at least two connectingcarbon atoms in a chain or ring. One class of diols compriseshydrocarbons substituted by exactly two hydroxy groups. One such diol ispinacol and another is pinanediol; there may also be mentionedneopentylglycol, 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol,2,3-butanediol, 1,2-diisopropylethanediol, 5,6-decanediol and1,2-dicyclohexylethanediol.

The alkyl groups of the dialkyl ether preferably have 1, 2, 3 or 4carbon atoms and the alkyl groups may be the same or different. Anexemplary ether is diethyl ether.

The alkyl groups of the dialkanolamine preferably have 1, 2, 3 or 4carbon atoms and the alkyl groups may be the same or different. Anexemplary dialkanolamine is diethanolamine. The diethanolamine/boronicacid reaction product hydrolyses in water at room temperature and therate of hydrolysis may be accelerated by adding acid or base.

The polar organic solvent is preferably CHCl₃. Other examples arepolyhalogenated alkanes generally and ethyl acetate. In principle, anypolar organic solvent is acceptable other than alcohols.

The aqueous acid is suitably a strong inorganic acid at a pH in theregion of 1 such as hydrochloric acid, for example.

After reaction with the acid, the reaction mixture is suitably washedwith, for example, NH₄Cl or another mild base.

An example of a specific procedure is as follows:

1. The pinacol or pinanediol ester of the selected peptide boronic acidis dissolved in diethyl ether.

2. Diethanolamine is added and the mixture is refluxed at 40° C.

3. The precipitated product is removed (filtered), washed (usuallyseveral times) with diethyl ether or another polar organic solvent otherthan an alcohol, and dried (e.g. by evaporation under vacuum).

4. The dry product is dissolved in a polar organic solvent other than analcohol, e.g. CHCl₃. Aqueous acid or base is added, e.g. hydrochloricacid (pH 1), and the mixture is stirred for e.g. approximately 1 h atroom temperature.

5. The organic layer is removed and washed with NH₄Cl solution.

6. The organic solvent is distilled off and the residual solid productis dried.

The above process results in the formation of what may conveniently bereferred to as a “diolamine adduct” of the peptide boronic acids offormula (I), especially such adducts with diethanolamine, and suchadducts are themselves included in the disclosure. The molecularstructure of such adducts is not known: they might comprise a compoundin which the two oxygens and the nitrogen of the diolamine are allcoordinated to the boron; they might comprise ions. The adducts arehowever considered to be esters. A particular novel product included inthe disclosure is that obtainable by reacting a pinacol or pinanediolester of a compound of Formula (IX), particularly (R,S,R)-TRI 50c, anddiethanolamine, i.e. the novel product is an (R,S,R)-TRI50c/diethanolamine “adduct” where the acid is (R,S,R)-TRI 50c.

The diolamine materials of the disclosure may be defined as acomposition of matter comprising:

-   -   (i) a species of formula (X)

wherein X is H or an amino protecting group, the boron atom isoptionally coordinated additionally with a nitrogen atom, and thevalency status of the terminal oxygens is open (they may be attached toa second covalent bond, be ionised as —O⁻, or have some other, forexample intermediate, status); and, in bonding association therewith

-   -   (ii) a species of formula (XI)

wherein the valency status of the nitrogen atom and the two oxygen atomsis open. It will be appreciated that the terminal oxygen atoms of thespecies of formula (X) and the oxygen atoms of the species of formula(XI) may be the same oxygen atoms, in which case the species of formula(XI) forms a diol ester with the species of formula (X).

It will be appreciated that the aforegoing technique comprises anexample of a method for recovering an organoboronic acid product, themethod comprising providing in a solvent a dissolved mixture comprisingthe organoboronic acid in a soluble form and a compound having twohydroxy groups and an amino group (i.e. a diolamine), causing orallowing the organoboronic acid and the diolamine to react to form aprecipitate, and recovering the precipitate. The soluble form of theorganoboronic acid may be a diol ester, as discussed above. The solventmay be an ether, as discussed above. The organoboronic acid may be oneof the organoboronic acids referred to in this specification, forexample it may be of Formula III. The method described in this paragraphis novel and forms an aspect of the disclosure. A recovery method isfiltration.

The reaction between the diolamine and the soluble form of theorganoboronic acid is suitable carried out at an elevated temperature,for example under reflux.

Another aspect of the present disclosure is a method for recovering anorganoboron species, comprising

-   -   providing, in a form soluble in an ether, an organoboronic acid,        for example a drug such as, e.g. , a compound of formula (III)        or (IV);    -   forming a solution of the soluble form in the ether;    -   combining the solution with a dialkanolamine and allowing or        causing the dialkanolamine to react with the soluble form of the        organoboronic acid to form an insoluble precipitate; and    -   recovering the precipitate.

The term “soluble” in the preceding paragraph refers to species whichare substantially more soluble in the reaction medium than is theprecipitated product. In variants of the method, the ether is replacedby toluene or another aromatic solvent.

The diethanolamine precipitation technique described above is an exampleof another novel method, which is a method for recovering from ethersolution a pinacol or pinanediol ester of a peptide boronic acid,comprising dissolving diethanolamine in the solution, allowing orcausing a precipitate to form and recovering the precipitate. Thedisclosure encompasses variants of this methods in which another diolthan pinacol or pinanediol is used.

The precipitated material, i.e. the “adduct”, may be converted into thefree organoboronic acid, for example by contacting it with an acid. Theacid may be an aqueous acid, for example an aqueous inorganic acid, e.g.as described above. The precipitate may be dissolved, for example in anorganic solvent, prior to being contacted with the acid.

The disclosure therefore provides a method for making an organoboronicacid, comprising converting its diolamine reaction product to the acid.

The acid resulting from the methods described in the previous twoparagraphs may be converted to a salt of the acid with a multivalentmetal, which salt may in turn be formulated into a pharmaceuticalcomposition in oral dosage form.

3. Salt Synthesis

In general, the salts may be prepared by contacting the relevant boronicacid with a relevant base, e.g. the metal hydroxide (alternatively,metal carbonates might be used, for example). Sometimes it is moreconvenient to contact the acid with a relevant metal alkoxide (e.g.methoxide), for which purpose the corresponding alkanol is a suitablesolvent. Illustrative salts are acid salts (one —BOH proton replaced)and, to make these salts, the acid and the base are usually reacted insubstantially the appropriate stoichiometric quantities. Generallystated, therefore, the usual acid:base molar ratio is substantially n:1,where n is the valency of the metal cation of the base.

In one procedure, a solution of the peptide boronic acid in awater-miscible organic solvent, for example acetonitrile or an alcohol(e.g. ethanol, methanol, a propanol, for example iso-propanol, oranother alkanol), is combined with an aqueous solution of the base. Theacid and the base are allowed to react and the salt is recovered. Thereaction is typically carried out at ambient temperature (e.g. at atemperature of from 15 to 30° C., e.g. 15 to 25° C.), but an elevatedtemperature may be used, for example up to the boiling point of thereaction mixture but more usually lower, e.g. a temperature of up to 40°C. or 50° C. The reaction mixture may be allowed to stand or be agitated(usually stirred).

The time during which the acid and the base are allowed to react is notcritical but it has been found desirable to maintain the reactionmixture for at least one hour. A period of from one to two hours isusually suitable but longer reaction times may be employed.

The salt may be recovered from the reaction mixture by any suitablemethod, for example evaporation or precipitation. Precipitation may becarried out by adding an excess of a miscible solvent in which the salthas limited solubility. In one preferred technique, the salt isrecovered by evacuating the reaction mixture to dryness. The salt ispreferably thereafter purified, for example by redissolving the saltbefore filtering the resulting solution and drying it, for example byevacuating it to dryness. The redissolution may be performed usingwater, e.g. distilled water. The salt may then be further purified, forexample in order to remove residual water by further redissolution in asuitable solvent, which is advantageously ethyl acetate or THF followedby evaporating to dryness. The purification procedure may be carried outat ambient temperature (say, 15 to 30° C., e.g. 15 to 25° C.), or at amodestly elevated temperature, such as e.g. a temperature not exceeding40° C. or 50° C.; for example the salt may be dissolved in water and/orsolvent by agitating with or without warming to, for example, 37° C.

Also included is a method for drying the salts of the disclosure andother peptide boronic acid salts, comprising dissolving them in anorganic solvent, e.g. ethyl acetate or THF, and then evaporating todryness, e.g. by evacuation.

Generally, preferred solvents for use in purifying the salts are ethylacetate or THF, or perhaps another organic solvent.

A general procedure for synthesising multivalent metal salts ofCbz-Phe-Pro-BoroMpg-OH is as follows:

Cbz-Phe-Pro-BoroMpg-OH (20.00 g, 38.1 mM) is dissolved in acetonitrile(200 ml) with stirring at room temperature. To this solution is addedthe requisite base as a solution in distilled water (190 ml) [0.1Msolution for a divalent metal; 0.67M solution for a trivalent metal].The resultant clear solution is allowed to react for example by beingleft to stand or being agitated, for a usual period, in either case, offrom one to two hours. The reaction is typically carried out at ambienttemperature (e.g. 15-30° C., e.g. 15 to 25° C.) but alternatively thetemperature may be elevated (e.g. up to 30° C., 40° C. or 50° C.). Thereaction mixture is then evacuated to dryness under vacuum with itstemperature not exceeding 37° C., typically to yield a white brittlesolid or an oil/tacky liquid. The oil/tacky liquid is redissolved in theminimum amount of distilled water necessary (200 ml to 4L), typicallywith warming (e.g. to 30-40° C.), usually for up to 2 hours. Thesolution is filtered, suitably through filter paper, and evacuated todryness, again with the temperature of the solution not exceeding 37°C., or freeze dried. The resultant product is dried under vacuumovernight to normally yield a white brittle solid. If the product ispresent as an oil or tacky solid then it is dissolved in ethyl acetateand evacuated to dryness to produce the product as a white solid. Thewhite solid is typically a coarse, amorphous powder.

In variations of the aforegoing general procedure, the acetonitrile isreplaced by another water-miscible organic solvent, notably an alcohol,as discussed above, especially ethanol, methanol, iso-propanol oranother propanol.

The above synthetic procedures are applicable also to preparing alkalimetal salts of TRI 50c and other boronic acids described herein, forexample those of formula (III). These alkali metal salts are useful as astarting material for alternative syntheses of multivalent metal salts,where direct synthesis from the acid is inconvenient, as in the case ofa multivalent metal hydroxide which is less soluble in a selectedreaction medium for salt formation (e.g. zinc hydroxide). When an alkalimetal salt is being made as starting material, the stoichiometry of thereaction used to make the alkali metal salt is usually adjusted to 1:1,in order to prepare an acid salt. In such an “indirect” synthesis froman alkali metal salt, especially the sodium salt or alternatively thepotassium salt, the boronate alkali metal salt in solution is contactedwith a salt of the relevant metal (normally a salt having apharmaceutically acceptable anion, e.g. chloride). The salt of the“target” metal (e.g. zinc) is typically used in a stoichiometry (boronicacid:target metal) of n:1, where n is the valency of the metal. Themultivalent metal salt of the boronic acid is then recovered, forexample it will often precipitate out (when the multivalent metal saltis less soluble in the reaction medium than is the alkali metal salt).The resulting precipitate may then be separated from the liquid, e.g. byfiltration, and purified.

The preparation of the multivalent metal salts from the correspondingalkali metal salts is novel. The alkali metal salts and their aqueoussolutions also form part of the present disclosure. The alkali metalsalts are an advantageous as compared to the corresponding acids in thatthey are more resistant to degradation (their boropeptide moieties areless prone to degrade than are those of the corresponding free acids).

Also provided is the use of a peptide boronic acid drug, for example athrombin inhibitor or an acid of formula (I), to make a salt asdisclosed herein. Included also is a method of preparing a product,comprising contacting a peptide boronic acid drug, e.g. of formula (I),(II), (III), (IV) or (IX), with a base capable of making such a salt.

The peptide boronic acid used to prepare the pharmaceutical preparationsis typically of GLP or GMP quality, or in compliance with GLP (goodlaboratory practice) or GMP (good manufacturing practice). Such acidsare included in the disclosure.

Similarly the acids are usually sterile and/or acceptable forpharmaceutical use, and one aspect of the present disclosure resides ina composition of matter which is sterile or acceptable forpharmaceutical use, or both, and comprises a peptide boronic acid offormula (IV). Such a composition of matter may be in particulate form orin the form of a liquid solution or dispersion.

The intermediate acid may be in isolated form and such isolated acidsare included in the present disclosure, especially isolated acids whichare a peptide boronic acid of formula (IX):

X—(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂   (IX)

wherein X is H (to form NH₂) or an amino-protecting group.

One typical way of providing the intermediate acids is as a particulatecomposition consisting predominantly of such a peptide boronic acid, andthese compositions are included in the disclosure. The peptide boronicacid often forms at least 75% by weight of the composition and typicallyat least 85% by weight of the composition, e.g. at least 95% by weightof the composition.

Another typical way of providing the intermediate acids is as a liquidcomposition consisting of, or consisting essentially of, a peptideboronic acid of formula (II) and a liquid vehicle in which it isdissolved or suspended. The liquid vehicle may be an aqueous medium,e.g. water, or an alcohol, for example methanol, ethanol, isopropanol,or another propanol, another alkanol or a mixture of the aforegoing.

The compositions of the intermediate acids are generally sterile. Thecompositions may contain the peptide boronic acid in finely dividedform, to facilitate further processing.

Separation of Stereoisomers

The stereoisomers of a peptide boronic ester or a synthetic intermediateaminoboronate may be resolved in, for example, any known way. Inparticular, stereoisomers of boronic esters may be resolved by HPLC.

EXAMPLES Examples 1 TO 3 Introductory Remarks

Apparatus

Throughout the following procedures of Examples 1 to 3, standardlaboratory glassware and, where appropriate, specialised apparatus forhandling and transferring of air sensitive reagents are used.

All glassware is heated at 140-160° C. for at least 4 hours before useand then cooled either in a desiccator or by assembling hot and purgingwith a stream of dry nitrogen.

Solvents

The organic solvents used in the procedures of Examples 1 to 3 are alldry. Suitably, they are dried over sodium wire before use.

Dryness

In the drying procedures of Example 1 to 3, products are tested fordryness (including dryness in terms of organic solvent) by observingweight loss on drying. The following procedure was followed to determineloss on drying: a sample was placed in a vacuum drier and dried at 40°C. at 100 mbar for 2 hours. Products are considered dry when thedecrease in weight upon drying is less than 0.5% of the total weight ofthe starting material.

Examples 1 to 3 describe performance of the following reaction schemeand conversion of the resultant TRI 50c to a calcium salt thereof:

Example 1 Synthesis of TRI 50d

Step 1: Z-DIPIN B

Procedure A

17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole) iodine and127 ml dry tetrahydrofuran are charged and heated to reflux. Then 15 mlof a solution of 66 g (608 mmole) 1-chloro-3-methoxypropane in 185 mldry tetrahydrofuran are added and stirred under reflux until thevigorous reaction starts. After the initial exotherm ceases, thesolution of 1-chloro-3-methoxypropane is added slowly to maintain gentlereflux until all the magnesium is consumed. After the reaction isfinished, the reaction mixture is cooled to ambient temperature andslowly added to a solution of 64.4 g (620 mmole) trimethylborate in 95ml dry tetrahydrofuran; the latter solution is cooled to below 0° C.and, if it warms up during the course of the reaction, the reactionmixture must be added to it sufficiently slowly to maintain thetemperature of this solution below 65° C. Upon complete addition, thereaction mixture is allowed to warm to about 0° C. and stirred foranother 60 minutes. Then a solution of 22.4 ml sulfuric acid in 400 mlwater is added slowly so as to maintain the temperature below 20° C. Thelayers are allowed to settle and the phases are separated. The aqueouslayer is rewashed three times with 200 ml tert.-butylmethylether. Thecombined organic layers are allowed to settle and additional waterseparated from this solution is removed. The organic layer is dried overmagnesium sulfate, filtered and evaporated to dryness. The evaporationresidue is filtered from the precipitated solid and the filtratedissolved in 175 ml toluene. 34.8 g (292 mmole) pinacol is charged tothe solution followed by stirring at ambient temperature for not lessthan 10 hours. The solution is evaporated to dryness, dissolved in 475ml n-heptane and washed three times with 290 ml saturated aqueoussolution of sodium hydrogen carbonate. The n-heptane solution isevaporated to dryness and the evaporation residue distilled and thefraction with Bp 40-50° C. at 0.1-0.5 mbar recovered.

Boiling point: 40-50° C./ 0.1-0.5 mbar

Yield: 40.9 g (70%) Z-DIPIN B (oil)

Procedure B

17.8 g (732.5 mmole) magnesium turnings, 0.1 g (0.4 mmole) iodine and127 ml dry tetrahydrofuran are charged and heated to reflux. Then 15 mlof a solution of 66 g (608 mmole) 1-chloro-3-methoxypropane in 185 mldry tetrahydrofuran are added and stirred under reflux until thevigorous reaction starts. After the initial exotherm ceases, thesolution of 1-chloro-3-methoxypropane is added slowly to maintain gentlereflux. After the reaction is finished, the reaction mixture is cooledto ambient temperature and slowly added to a solution of 64.4 g (620mmole) trimethylborate in 95 ml dry tetrahydrofuran, maintaining thetemperature of this solution below minus 65° C. Upon complete addition,the reaction mixture is allowed to warm to about 0° C. and stirred foranother 60 minutes. Then a solution of 22.4 ml sulfuric acid in 400 mlwater is added slowly so as to maintain the temperature below 20° C. Theorganic solvent is removed by distillation under vacuum. 300 mln-heptane is charged to the aqueous solution of the evaporation residuefollowed by addition of 34.8 g (292 mmole) pinacol. Thetwo-phase-mixture is stirred at ambient temperature for not less than 2hours. After allowing the layers to settle, the aqueous phase isseparated. 300 ml n-heptane is charged to the aqueous solution and thetwo-phase-mixture is stirred at ambient temperature for not less than 2hours. After allowing the layers to settle, the aqueous phase isseparated. The organic layers are combined and washed once with 200 mlwater, followed by 200 ml saturated sodium hydrogen carbonate solutionand two further washes with 200 ml water each. The n-heptane solution isevaporated to dryness and the evaporation residue distilled and thefraction with Bp 40-50° C. at 0.1-0.5 mbar recovered.

Boiling point: 40-50° C./0.1-0.5 mbar

Yield: 40.9 g (70-85%) Z-DIPIN B (oil)

Step 2: Z-DIPIN C

16.6 g (164 mmole) diisopropylamine and 220 ml tetrahydrofuran arecharged and cooled to −30 to −40° C. To this solution 41.8 g (163 mmole)n-butyl lithium, 25% in n-heptane is added, followed by stirring at 0 to−5° C. for one hour. This freshly prepared solution of lithiumdiisopropylamide is cooled to −30° C. and then added to a solution of27.9 g (139 mmole) Z-DIPIN B in 120 ml tetrahydrofuran and 35.5 g (418mmole) dichloromethane at a temperature between −60 and −75° C. Thesolution is stirred at that temperature for half an hour followed byaddition of 480 ml (240 mmole) 0.5N anhydrous Zinc(II)-chloride intetrahydrofuran or 32.5 g (240 mmole) anhydrous solid Zinc(II)-chloride.After stirring at −65° C. for one hour, the reaction mixture is allowedto warm to ambient temperature and stirred for another 16-18 hours. Thereaction mixture is evaporated to dryness (i.e. until solvent isremoved) and followed by addition of 385 ml n-heptane. The reactionmixture is washed with 150 ml 5% sulfuric acid, with 190 ml saturatedsodium hydrogen carbonate solution, and 180 ml saturated sodium chloridesolution. The organic layer is dried over magnesium sulfate, filteredand evaporated to dryness (i.e. until solvent is removed). The oilyresidue is transferred into the next step without further purification.

Yield: 19 g (55%) Z-DIPIN C

Step 3: Z-DIPIN D

To a solution of 23.8 g (148 mmole) hexamethyidisilazane in 400 mltetrahydrofuran at −15° C. is added 34.7 g (136 mmole) n-butyl lithium,25% in n-heptane and stirred for one hour. The solution is cooled to−55° C. followed by the addition of 30.6 g (123 mmole) Z-DIPIN Cdissolved in 290 ml tetrahydrofuran and 35 ml tetrahydrofuran to thisfreshly prepared solution of LiHMDS. The solution is allowed to warm toambient temperature and stirred for 12 hours. The reaction mixture isevaporated to dryness, the evaporation residue dissolved in 174 mln-heptane, washed with 170 ml water and 75 ml saturated sodium chloridesolution. The organic phase is dried over magnesium sulfate, filteredand evaporated to complete dryness (i.e. until solvent is removed). Theoily residue is dissolved in 100 g n-heptane. This solution is carriedover into the next step without further purification.

Yield: 32.2 g (70%) Z-DIPIN D

Step 4: Z-DIPIN (TRI50b, crude)

A solution of 26.6 g (71 mmole) Z-DIPIN D in 82.6 g n-heptane is dilutedwith 60 ml n-heptane and cooled to −60° C. followed by introduction of10.5 g (285 mmole) hydrogen chloride. The reaction mixture issubsequently evacuated and flushed with nitrogen, while the temperatureis increased in increments of about 20° C. to ambient temperature. Thesolvent is removed from the oily precipitate and replaced several timesby 60 ml fresh n-heptane. The oily residue is dissolved in 60 mltetrahydrofuran (Solution A).

To a different flask 130 ml tetrahydrofuran, 24.5 g (61.5 mmole)Z-D-Phe-Pro-OH and 6.22 g (61.5 mmole) N-methylmorpholine are chargedand cooled to −20° C. To this solution a solution of 8.4 g (61.5 mmole)isobutylchloroformate in 20 ml tetrahydrofuran is added and stirred for30 minutes, followed by addition of Solution A at −25° C. Upon completeaddition, up to 16 ml (115 mmole) triethylamine is added to adjust thepH to 9-10, measured using a pH stick. The reaction mixture is allowedto warm to ambient temperature and stirred for 3 hours, still undernitrogen. The solvent is evaporated to dryness and the evaporationresidue dissolved in 340 ml tert.-butylmethylether (t-BME). The solutionof Z-DIPIN in t-BME is washed twice with 175 ml 1.5% hydrochloric acid.The combined acidic washes are given a rewash with 175 ml t-BME. Thecombined organic layers are washed with 175 ml water, with 175 mlsaturated sodium hydrogen carbonate solution, with 175 ml 25% sodiumchloride solution, dried over magnesium sulfate and filtered. Thissolution is carried over into the next step without furtherpurification.

Yield: 29.9 g (80%) Z-DIPIN

Example 2 Synthesis of TRI 50d (Diethanolamine Adduct of TRI 50c)

The starting material used in this Example is the solution of TRI 50b(“Z-DIPIN”) obtained in Example 1. The solution is carried forward tothe synthesis of TRI 50d without further purification. The solution ofZ-DIPIN in t-BME (containing 7.0 g (11.5 mmole) (R,S,R) TRI50b,calculated based on HPLC results of Z-DIPIN) is evaporated to drynessand the evaporation residue dissolved in 80 ml diethylether. 1.51 g(14.4 mmole) diethanolamine is added and the mixture heated at refluxfor at least 10 hours, during which process the product precipitates.The suspension is cooled to 5-10° C., filtered and the filter residuewashed with diethylether.

To improve chiral and chemical purity the wet filter cake (7 g) isdissolved in 7 ml dichloromethane, cooled to 0-5° C. and the productprecipitated by addition of 42 ml diethylether and filtered. Theisolated wet product is dried at 35° C. in vacuum or at least 4 hours,until day.

Yield: 5.5 g (80%) Tri50d

Melting Point: 140-145° C.

Example 3 Preparation of Calcium Salt of TRI50c

1.5 kg (2.5 mole) TRI50d from Example 2 is dissolved in 10.5 Ldichloromethane. 11 L 2% hydrochloric acid is added and the mixture isstirred for at most 30 minutes (optimally about 20 minutes) at roomtemperature. After stirring the layers are allowed to settle andseparated. The aqueous layer is given a rewashed twice with 2.2 Ldichloromethane. The combined organic layers are washed with a solutionof 625 g ammonium chloride in 2.25 L water. The organic phase is driedover magnesium sulfate, filtered and the filtrate evaporated to dryness.An assay of the free boronic acid is performed and the amounts of thesolvents and base for conversion of the acid to the salt are calculated.If 2.5 mol of the free acid is obtained, the evaporation residue isdissolved in 5 L acetonitrile followed by addition of a suspension of 93g (1.25 mole) calcium hydroxide in 1 L water. The solution is stirredfor two hours at ambient temperature (e.g. 15-30° C., optimally roomtemperature) and then evaporated under vacuum (of ca. 10 mmHg) at atemperature initially of about 10° C. and then increasing to a limit ofabout 35° C. The evaporation residue is repeatedly dissolved in 3.5 Lfresh acetonitrile and evaporated to dryness to remove traces of water.If the evaporation residue is dry, it is dissolved in 6 Ltetrahydrofuran and slowly added to a mixture of 32 L n-heptane and 32 Ldiethylether. The addition is performed slowly enough to avoid lumpingor sticking of the product and is carried out over a period of not lessthan 30 minutes. The precipitated product is filtered off, washed withn-heptane and dried under vacuum (of ca. 10 mmHg) at a temperature below35° C. until dry.

Yield: 0.98 kg (70%) Tri50c calcium salt.

The procedures of Examples 1 to 4 may be scaled up and, if operatedcarefully, will produce highly pure salts. In the diethanolamineprecipitation step it is important to use 1.25 equivalents ofdiethanolamine per equivalent of (R,S,R) TRI 50b. In the hydrolysis ofthe diethanolamine ester, it is important to avoid excessively longcontact with the aqueous acid. Likewise the TRI 50b should besynthesised via the Grignard reaction to Z-DIPIN A.

Example 4 Alternative Conversion of TRI 50b to TRI 50c

The synthetic procedures described in this and subsequent syntheticexamples were generally performed under nitrogen and using dry solventsas supplied from commercial sources.

1. Approximately 300 g of TRI 50b, obtained by the HPLC purification ofracemic TRI 50b) were dissolved in approximately 2.5 L diethylether. Itis estimated that different batches of TRI 50b had isomeric puritiesranging from 85% R,S,R to in excess of 95% R,S,R.

2. Approximately 54 ml diethanolamine were added (1:1 stoichiometry withtotal TRI 50b content), and the mixture was refluxed at 40° C.

3. The precipitated product was removed, washed several times withdiethylether and dried.

4. The dry product was dissolved in CHCl₃. Hydrochloric acid (pH 1) wasadded and the mixture was stirred approximately 1 h at room temperature.

5. The organic layer was removed and washed with NH₄Cl solution.

6. The organic solvent was distilled off and the residual solid productwas dried.

Typical yield: Approximately 230 g

Example 5 First Alternative Preparation of Calcium of TRI 50c

Cbz-Phe-Pro-BoroMpg-OH (20.00 g, 38.1 mM) obtained by the method ofExample 4 is dissolved in acetonitrile (200 ml) with stirring at roomtemperature. To this solution is added Ca(OH)₂ as a 0.1M solution indistilled water (190 ml). The resultant clear solution is stirred for 2hours at room temperature and then evacuated to dryness under vacuumwith its temperature not exceeding 37° C. The resultant product is awhite brittle solid.

The salt was then dried under vacuum over silica to constant weight (72h).

Yield: 17.69 g.

Example 6 Second Alternative Preparation of Calcium Salt of TRI 50c

50.0 g TRI 50c (95.2 mmol) were dissolved under stirring in 250 mlacetonitrile at room temperature and then cooled with an ice bath. Tothis ice cooled solution 100 ml of an aqueous suspension of 3.5 g (47.6mmol) calcium hydroxide was added dropwise, stirred for 2.5 hours atroom temperature, filtered and the resulting mixture evaporated todryness, the temperature not exceeding 35° C. The clear yellowish oilyresidue was redissolved in 200 ml acetone and evaporated to dryness. Theprocedure of redissolving in acetone was repeated one more time toobtain colourless foam.

This foam was redissolved in 100 ml acetone, filtered and added dropwiseto an ice cooled solution of 1100 ml petrol ether 40/60 and 1100 mldiethylether. The resulting colourless precipitate was filtered, washedtwo times with petrol ether 40/60 and dried under high vacuum, yielding49.48 g of a colourless solid (92%), with a purity of 99.4% according toan HPLC measurement.

Example 7 UV/Visible Spectra of Calcium Salt of TRI 50c

UV/Visible spectra of the salt resulting from the procedure of Example 5were recorded in distilled water at 20° C. from 190 nm to 400 nm. TRI50C and the salt gave λ_(max) at 210 and 258 nm. The weight of the driedsalt was then measured for the purposes of calculating the extinctioncoefficient. The λ_(max) at 258 nm was used. The extinction coefficientwas calculated using the formula:

A=εcl where A is the absorbance

-   -   C is the concentration    -   I the path length of the UV cell    -   and ε is the extinction coefficient.

Extinction coefficient: 955.

Example 8 Aqueous Solubility of Calcium Salt of TRI 50c

The salt used in this Example was made using a modification of theprocess described in Example 6. The modified process differs from thatdescribed in that 100 mg of TRI 50c was used as starting material, theproduct of the redissolution in water was dried by freeze drying and thefiltration was carried out through a 0.2 μm filter. The salt is believedto contain about 85% of R,S,R isomer.

To determine maximum aqueous solubility 25 mg of the dried salt wereshaken in water at 37° C., the sample filtered and the UV spectrummeasured. The salt left a white residue of undissolved material.

Solubility when dissolved at 25 mg/ml: 5 mM (5 mg/ml).

Example 9 In Vitro Activity of Calcium Salt of TRI 50c

TRI 50c calcium salt was assayed as an inhibitor of human α-thrombin byan amidolytic assay (J. Deadman et al, J. Med. Chem. 38:15111-1522,1995, which reports a Ki value of 7 nM for TRI 50b).

The inhibition of human α-thrombin therefore, was determined by theinhibition of the enzyme catalysed hydrolysis of three differentconcentrations of the chromogenic substrate S-2238.

200 μl of sample or buffer and 50 μl of S-2238 were incubated at 37° C.for 1 minute and 50 μl of human α-thrombin (0.25 NIHμ/ml) was added. Theinitial rate of inhibited and uninhibited reactions were recorded at 4.5nm. The increase in optical density was plotted according to the methodof Lineweaver and Burke. The Km and apparent Km were determined and Kiwas calculated using the relationship.

$V = \frac{V\; \max}{1 + {\frac{Km}{\lbrack S\rbrack} \cdot \left( {1 + \frac{\lbrack I\rbrack}{Ki}} \right)}}$

The buffer used contained 0.1M sodium phosphate, 0.2M NaCl, 0.5% PEG and0.02% sodium azide, adjusted to pH 7.5 with orthophosphoric acid.

The samples consist of the compound dissolved in DMSO.

The reader is referred to Dixon, M and Webb, E. C., “Enzymes”, thirdedition, 1979, Academic Press, the disclosure of which is incorporatedherein by reference, for a further description of the measurement of Ki.

TRI 50c calcium salt was observed to have a Ki of 10 nM.

Example 10 Preparation of Zinc Salt of TRI 50c

The relative solubilities of the respective hydroxides of magnesium andzinc are such that, if these hydroxides had been used to prepare thecorresponding TRI 50c salts using the procedure of Example 5, they wouldnot have resulted in homogeneous salt formation. New methods weretherefore developed to prepare the zinc and magnesium salts, asdescribed in this and the next examples.

TRI 50c sodium salt (2.24 g, 4.10 mM) was dissolved in distilled water(100 ml) at room temperature and zinc chloride in THF (4.27 ml, 0.5M)was carefully added with stirring. A white precipitate that immediatelyformed was filtered off and washed with distilled water. This solid wasdissolved in ethyl acetate and washed with distilled water (2×50 ml).The organic solution was evacuated to dryness and the white solidproduced dried over silica in a desiccator for 3 days beforemicroanalysis. Yield 1.20 g.

¹H NMR 400 MHz, δ_(H)(CD₃OD) 7.23-7.33 (20H, m, ArH), 5.14 (4H, m,PhCH₂O), 4.52 (4H, m, αCH), 3.65 (2H, m), 3.31 (12H, m), 3.23 (6H, s,OCH₃), 2.96 (4H, d, J7.8 Hz), 2.78 (2H, m), 2.58 (2H, m), 1.86 (6H, m),1.40 (10H, m).

¹³C NMR 75 MHz 393K δ_(C)(CD₃OD) 178.50, 159.00, 138.05, 137.66, 130.54,129.62, 129.50, 129.07, 128.79, 128.22, 73.90, 67.90, 58.64, 58.18,56.02, 38.81, 30.06, 28.57, 28.36, 25.29. FTIR (KBr disc) ν_(max) (cm⁻¹)3291.1, 3062.7, 3031.1, 2932.9, 2875.7, 2346.0, 1956.2, 1711.8, 1647.6,1536.0, 1498.2, 1452.1, 1392.4, 1343.1, 1253.8, 1116.8, 1084.3, 1027.7,916.0, 887.6, 748.6, 699.4, 595.5, 506.5.

Example 11 Preparation of Magnesium Salt of TRI 50c

TRI 50c (1.00 g, 1.90 mM) was dissolved in methanol (10 ml) and stirredat room temperature. To this solution was added magnesium methoxide(Mg(CH₃O)₂) in methanol (1.05 ml, 7.84 wt %). This solution was stirredfor 2 hours at room temperature filtered and evacuated to 5 ml. Water(25 ml) was then added and the solution evacuated down to dryness toyield a white solid. This was dried over silica for 72 hours beforebeing sent for microanalysis. Yield 760 mg.

¹H NMR 300 MHz, δ_(H)(CD₃C(O)CD₃) 7.14-7.22 (20H, m), 6.90 (2H, m), 4.89(4H, m, PhCH₂O), 4.38 (2H, m), 3.40 (2H, br s), 2.73-3.17 (20H, broadunresolved multiplets), 1.05-2.10 (16H, broad unresolved multiplets).

¹³C NMR 75 MHz 393K δ_(C)(CD₃C(O)CD₃) 206.56, 138.30, 130.76, 129.64,129.31, 129.19, 129.09, 128.20, 128.04, 74.23, 73.55, 67.78, 58.76,56.37, 56.03, 48.38, 47.87, 39.00, 25.42, 25.29. FTIR (KBr disc) ν_(max)(cm⁻¹) 3331.3, 3031.4, 2935.3, 2876.9, 2341.9, 1956.1, 1711.6, 1639.9,1534.3, 1498.1, 1453.0, 1255.3, 1115.3, 1084.6, 1027.6, 917.3, 748.9,699.6, 594.9, 504.5, 467.8.

Example 12 Analysis of Calcium, Magnesium and Zinc Salts of (R,S,R) TRI50c

The following salts were prepared using a boronate:metal stoichiometryof n:1, where n is the valency of the metal, using (R,S,R) TRI 50c ofhigher chiral purity than that used to prepare the salts described inExample 8.

A. Calcium Salt (Product of Example 5)

Analytical data HPLC or LC/MS: HPLC betabasic C18 Column, CH₃CN, WaterEstimated Purity: >95% by UV (λ_(215 nm)) Micro analysis: Calcd. Found.C: 59.27 55.08 H: 6.48 6.43 N: 7.71 7.08 Other: B: 1.99 2.01 Ca: 3.683.65 Physical Properties Form: Amorphous solid Colour: White MeltingPoint: N/A Solubility: Soluble in aqueous media ca ~4 mg/ml M_(w):1088.89

B. Magnesium Salt (Product of Example 11)

Analytical data HPLC or LC/MS: HPLC betabasic C18 Column, CH₃CN, WaterEstimated Purity: >90% by UV (λ_(215 nm)) Micro analysis: Cal/cd. Found.C: 60.44 57.25 H: 6.57 6.71 N: 7.83 7.45 Other: B: 2.01 2.02 Mg: 2.262.12 Physical Properties Form: Amorphous solid Colour: White MeltingPoint: N/A Solubility: Soluble in aqueous media ca~7 mg/ml M_(w):1073.12

C. Zinc Salt (Product of Example 10)

Analytical data HPLC or LC/MS: HPLC betabasic C18 Column, CH₃CN, WaterEstimated Purity: >95% by UV (λ_(215 nm)) Micro analysis: Cal/cd. Found.C: 58.21 56.20 H: 6.33 6.33 N: 7.54 7.18 Other: B: 1.94 1.84 Zn: 5.877.26 Physical Properties Form: Amorphous solid Colour: White MeltingPoint: N/A Solubility: Soluble in aqueous media ca~2 mg/ml M_(w):1114.18

Notes: The trigonal formula of the acid boronate is used in thecalculated microanalyses. It is believed that a lower calcium saltsolubility is reported in example 8 because the salt tested in example 8had lower chiral purity.

Conclusion

The zinc, calcium and magnesium salts have all been prepared with astoichiometry of one metal ion to two molecules of TRI 50c. The valuesfound for the calcium and magnesium salts are close to and thusconsistent with those calculated for this 1:2 stoichiometry. For thezinc salt an excess of zinc was found; nonetheless, the zinc saltcomprises a significant proportion of acid boronate.

Example 13 Stability

This Example compares the stability of TRI 50c and TRI 50c calcium saltwhen filled into enteric-coated hard gelatin capsules (see Example 20).

1. Tabulated Results

Climatic Purity (HPLC Purity (HPLC conditions % Area) % Area)³ Compound.Packing 1.5 month⁰⁾ T0 T1 TRI50c capsules 25° C./60% 99 73.9 in blisterr.h.⁴ TRI50c capsules 40° C./75% 99 73.9 in blister r.h TRI50c capsules¹40° C./75% 99 75.3 r.h TRI50c capsules 25° C./60% 99.2²⁾ 98.0 CalciumSalt in blister r.h. TRI50c capsules 40° C./75% 99.2² 97.2 Calcium Saltin blister r.h TRI50c capsules¹ 40° C./75% 99.2² 95.0 Calcium Salt r.hNotes: ⁰⁾1.5 month storage at given conditions, samples were then storedat room temperature until analytical testing. ¹capsules stored at therespective climatic conditions without blister. ²⁾purity of the batchbefore storage. ³purity of the stored batch (capsules were poured out,the contents of the capsules were then analyzed). ⁴r.h. = relativehumidity

2. Analytical Procedure

2.1 Sample Preparation

2.1.1 Assay of TRI 50c and Salts

TRI 50c-standard (free acid) was stored in a desiccator over phosphoruspentoxide for 2 days for drying. Afterwards, the reference standard wasweighed in a volumetric flask and dissolved in a mixture of acetonitrileand water (25/75 v/v %). Aliquots of the resulting solution (ST 1A) werediluted successively with water as shown in the dilution scheme of table4.

Stock- and Calibration solutions of Tri 50c. Net weight Purity DissolvedConc. Calibr. mg % Salt-Factor in ml Solvent [μg/ml] [μg/ml] ST 1A 40.898.23 1 10 ACN/water 4007.8 C4000 25/75 (v/v %) ml ST [μg/ml] ad mlSolvent [μg/ml] ST 2A 5 1A 4007.8 10 water 2003.9 C2000 ST 3A 5 2A2003.9 10 water 1001.9 C1000 ST 4A 5 3A 1001.9 10 water 501.0 C500 ST 5A5 4A 500.9 10 water 250.5 C250 ST 6A 1 3A 1001.9 10 water 100.2 C100 ST7A 1 6A 100.2 10 water 10.0 C10

2.1.2 Impurity Profile of the Stored Capsules

The stored capsules of every batch at corresponding climatic conditionwere removed and 10 mg of the content was weighed in a 10 ml volumetricflask and dissolved in 10 ml of a mixture of acetonitrile/water (25/75v/v %). These solutions were injected for impurity profile analysis andfor quantification respectively.

3. Data Evaluation

The quantitative evaluation and the impurity profile analysis wereperformed using an HPLC-PDA method. The processing wavelength was set as258 nm.

4. Analytical Parameters

4.1 Equipment and Software

Autosampler Waters Alliance 2795 Pump Waters Alliance 2795 Column ovenWaters Alliance 2795 Detection Waters 996 diode array, extractedwavelength 258 nm Software version Waters Millennium Release 4.0

4.2 Stationary Phase

Analytical Column ID S71 Material X-Terra ™ MS C_(18,) 5 μm SupplierWaters, Eschborn, Germany Dimensions 150 mm × 2.1 mm (length, internaldiameter)

4.3 Mobile Phase

Aqueous phase: A: 0.1% HCOOH in water

Organic phase: C: ACN

Gradient conditions:

Time Flow % A % C 0.00 0.5 90 10 27.0 0.5 10 90 27.1 0.5 90 10 30.0 0.590 10

5. Impurity Profile Tables of TRI50C Ca Salt

Capsules in blister 25° C./60% r.h. Amount Retention Time Name [μg/ml][min.] Area % Area Height Benzaldeh. 6,058 7927 1.29 392 Tri50c 930,90311,686 601551 98.02 25135 19,199 839 0.14 89 19,498 1821 0.30 105 20,1681581 0.26 158

The corresponding HPLC trace is shown in FIG. 1.

Capsules in blister 40° C./75% r.h. Amount Retention Time Name [μg/ml][min.] Area % Area Height Benzaldeh. 6,060 12270 2.37 586 Tri50c 786,22311,681 503867 97.19 21324 19,517 707 0.14 97 20,185 1614 0.31 169

The corresponding HPLC time is shown in FIG. 2.

Capsules (no blister) 40° C./75% r.h. Amount Retention Time Name [μg/ml][min.] Area % Area Height Benzaldeh. 6,041 19170 3.64 992 Imp. I 10,8974433 0.84 345 Tri50c 780,097 11,666 499730 94.96 21526 19,494 805 0.15110 20,156 2100 0.40 176

The corresponding HPLC trace is shown in FIG. 3.

Example 14 In-Vitro Assay as Thrombin Inhibitor of Magnesium Salt of TRI50c

Thrombin Amidolytic Assay

TRI 50c magnesium salt (TRI 1405) was tested in a thrombin amidolyticassay.

Reagents:

Assay Buffer:

100 mM Na phosphate

200 mM NaCl (11.688 g/l)

0.5% PEG 6000 (5 g/l)

0.02% Na azide

pH 7.5

Chromogenic substrate S2238 dissolved to 4 mM (25 mg+10 ml) in water.Diluted to 50 uM with assay buffer for use in assay at 5 μM. (S2238 isH-D-Phe-Pip-Arg-pNA).

Thrombin obtained from HTI, via Cambridge Bioscience, and aliquoted at 1mg/ml with assay buffer. Dilute to 100 ng/ml with assay buffer and thena further 1 in 3 for use in the assay.

Assay:

110 μl assay buffer

50 ul 5 μg/ml thrombin

20 μl vehicle or compound solution

5 minutes at 37° C.

20 μl 50 μM S2238

Read at 405 nm at 37° C. for 10 minutes and record Vmax

Results:

The results are presented in FIG. 4.

Discussion:

In this assay the magnesium salt of TRI 50c shows the same activity asTRI 50b as an external control.

Example 15 (Comparative)—Preparation of Potassium Salt of TRI 50c

Cbz-Phe-Pro-BoroMpg-OH (20.00 g, 38.1 mM) is dissolved in acetonitrile(200 ml) with stirring at room temperature. To this solution is addedKOH as a 0.2M solution in distilled water (190 ml). The resultant clearsolution is stirred for 2 hours at room temperature and then evacuatedto dryness under vacuum with its temperature not exceeding 37° C. Theresultant oil/tacky liquid is redissolved in 1 L distilled water withwarming to 37° C. for about 2 hours. The solution is filtered throughfilter paper and evacuated to dryness, again with the temperature of thesolution not exceeding 37° C. The resultant product is dried undervacuum overnight to normally yield a white brittle solid.

Yield: 14.45 mg.

The salt was then dried under vacuum over silica to constant weight (72h).

Microanalysis:

C % Found H % Found N % Found B % Found Metal % Found (Calc.) (Calc.)(Calc.) (Calc.) (Calc.) 54.84 6.25 7.02 2.01 K 4.29 (57.55) (6.26)(7.45) (1.92) (6.94)

Example 16 (Comparative)—Aqueous Solubility of Potassium Salt of TRI 50c

The UV/visible spectra of TRI 50c and its solubility were obtained asdescribed above in relation to the calcium salt. Solubility whendissolved at 25 mg/ml: 29 mM (16 mg/ml).

Example 17 (Comparative)—Solubility of TRI 50c

The UV/visible spectra of TRI 50c and its solubility were obtained asdescribed above in relation to the calcium salt. The solubility of TRI50c when dissolved at 50 mg/ml was 8 mM (4 mg/ml).

Example 18 Intraduodenal Absorption in Rat

A. Preparation of Liquid Formulations of TRI 50c and Salt

1. Preparation of Buffer Solution pH 4.5

Place 1.48 g of sodium acetate (anhydrous) in a 1000 mL volumetricflask, add 16 mL 2N CH₃COOH, then add water and mix. Adjust the pH to4,5 using 0.2 N NaOH and fill up with water.

2. Preparation of Buffer Solution pH 6.8 (USP)

Place 50.0 mL monobasic potassium phosphate 0.2 M in a 200 mL volumetricflask add 22.4 mL NaOH 0.2 M fill up with dest. Water. Check the pH andadjust if necessary.

3. Preparation of the Formulation

-   -   Place 10 mg of the relevant compound in an Eppendorf cup    -   Add 0.5 mL ethanol and shake for 10 minutes    -   Sonicate for 10 minutes    -   Add 1.5 mL of buffer    -   Shake for additional 15 minutes    -   Resulting target concentration: 5 mg/mL

B. Intraduodenal Studies

The intraduodenal studies were performed using male Wistar rats,approximately 8 weeks of age and weighing between 250 and 300 g.

Food was withheld overnight prior to dosing and returned approximately 2hours post-dose. Water was available ad libitum.

Animals were anaesthetised using gaseous halothane. A small incision wasmade in the abdomen and the duodenum located. Each animal received asingle administration of control or test article by injection directlyinto the duodenum, using a constant dose volume of 4 mL/kg. Followingadministration the incision was closed using surgical staples.

Individual dose volumes were based on individual body weights, obtainedon the day of dosing. Treatments employed for the study were as follows:

Formulation Dose level concentration Number of Treatment (mg/kg) (mg/mL)animals TRI 50c control 20 5 5 Calcium salt 20 5 5 Potassium saltcomparator 20 5 5

Approximately 0.6 mL of blood was collected via a tail vein into 3.8%tri sodium citrate tubes approximately 48 hours prior to dosing andagain at 0.5, 1, 2, 4 and 8 hours post-dose.

Plasma was prepared by centrifugation at 300 rpm for 10 minutes at 4° C.Plasma was stored frozen (nominally −20° C.) prior to analysis in anautomated coagulometer.

C. Results

TABLE 2 Mean thrombin time for intraduodenally dosed rats Dose Groupmean thrombin time (s ± sd) at time (hour) Treatment (mg/kg) −48 0.5 1 24 8 TRI 50c 20 21.3 ± 2.69  42.1 ± 19.54 27.5 ± 9.42 23.5 ± 6.40 21.8 ±2.33 21.5 ± 2.67 control Calcium salt 20 21.6 ± 1.77 42.0 ± 6.74 34.0 ±1.89 22.6 ± 5.10 24.4 ± 2.41 22.4 ± 1.73 Potassium salt 20 20.0 ± 1.9226.5 ± 3.64 24.4 ± 3.35 23.2 ± 0.83 23.2 ± 2.36 21.6 ± 0.70 comparatorsd = standard deviation

Example 19 Oral Absorption in Rat

A. Preparation of Liquid Formulations of TRI 50c and Salt

The procedure of Example 18 was followed.

B. Oral Studies

The per-oral studies were performed using male Wistar rats,approximately 8 weeks of age and weighing between 250 and 300 g.

Food was withheld overnight prior to dosing and returned approximately 2hours post-dose. Water was available ad libitum.

Each animal received a single administration of control or test articleby oral gavage, using a constant dose volume of 4 mL/kg.

Individual dose volumes were based on individual body weights, obtainedon the day of dosing.

Treatments employed for the study were as follows:

Formulation Dose level concentration Number of Treatment (mg/kg) (mg/mL)animals TRI 50c control 20 5 5 Calcium salt 20 5 5 Potassium saltcomparator 20 5 5

Approximately 0.6 mL of blood was collected via a tail vein into 3.8%tri sodium citrate tubes approximately 48 hours prior to dosing andagain at 0.5, 1, 2, 4 and 8 hours post-dose.

Plasma was prepared by centrifugation at 300 rpm for 10 minutes at 4° C.Plasma was stored frozen (nominally −20° C.) prior to analysis in anautomated coagulometer.

C. Results

TABLE 3 mean thrombin times in the rat following oral administrationDose Group mean thrombin time (s ± sd) at time (hour) Treatment (mg/kg)−48 0.5 1 2 4 8 TRI 50c 20 22.9 ± 2.28 26.8 ± 1.96 23.3 ± 3.68 23.9 ±2.25 23.1 ± 2.70 25.1 ± 0.33 control Calcium salt 20 23.4 ± 1.25 25.9 ±3.05 25.7 ± 1.94 24.3 ± 0.98 25.0 ± 1.31 22.9 ± 3.46 Potassium salt 2022.0 ± 1.40 24.7 ± 2.18 24.1 ± 1.87 22.9 ± 3.29 23.2 ± 1.24 23.8 ± 1.79comparator sd = standard deviation

Example 20 Intraduodenal Variation

The thrombin times determined in example 18 were analysed to determinethe standard deviation for increase in thrombin time, expressed as apercentage of the mean value (this is sometimes called the ‘coefficientof variation’). The variation for the Ca salt was calculated to be lessthan for TRI 50c, as shown in Table 4 below.

TABLE 4 Thrombin times in rats dosed intraduodenally

Conclusion

Examples 18 and 19 indicate that multivalent metal salts of boronicacids have a high oral bioavailability involving an unknown technicaleffect not linked to solubility.

Example 20 indicates that multivalent metal salts of boronic acids havea low variation in oral bioavailability involving an unknown technicaleffect not linked to solubility.

It is speculated that the technical effects may in some way involvecoordination between the boronate group and the metal ion.

Example 21 Oral Administration in Dog

The pharmacokinetics (PK) and pharmacodynamics (PD) of TRI 50c (freeacid) and its calcium salt were studied in beagle dogs following oraladministration. Three female and three male dogs were used for each legof the study. The weight range of the dogs was 8-18 kg.

The PD was measured as thrombin time and APTT using an automatedcoagulometer. Plasma concentrations were measured using an LCMS/MSmethod.

The calcium salt and TRI 50c were filled into gelatine capsules andenterically coated (HPMCP 55). The dose was tailored on an individualbasis for each dog. Blood samples were taken into tri-sodium citrate aspreviously at pre dose, 0.5, 1, 1.5, 2, 3, 6, 8, 12, 16 and 24 hourspost dose.

A. Results

A.1 Tolerance

The TRI 50c and the calcium salt were both tolerated well with noadverse events for the total duration of the study.

A.2 Calcium Salt

Unexpectedly high mean thrombin-clotting times were noted in dogsreceiving the calcium salt. C max was observed three hours post dosewith a mean thrombin clotting time of 80.5 seconds (raised from a baseline of 15 seconds). There was still elevation of mean thrombin clottingtimes 8 hours post dose (mean of 20.2 seconds). All dogs respondeddynamically following oral administration of the calcium salt, althoughthere was some variability in response. All dogs dosed with the calciumsalt achieved peak thrombin clotting times of up to 148 seconds,although the majority of animals (four out of six) achieved at least afour times elevation in peak thrombin time.

A.3 TRI50c

Absorption as estimated by examination of dynamic response (TT) wasvariable. A peak thrombin time was noted 1.5 hours post dose (34.2seconds from a base line of 15.4 seconds). Two animals failed tosignificantly absorb TRI 50c as estimated from their dynamic responses.

B. Activated Partial Thromboplastin Times

There were no significant changes in APTI from base line followingadministration of TRI 50c. There was a very slight mean elevation inAPTT at 3 hours following administration of the calcium salt (14.5seconds to 18 seconds at peak) this rise was deemed not to be clinicallyrelevant.

C. Bioavailability

An estimation of bioavailability was achieved by a conversion ofthrombin clotting times following administration of the calcium salt toestimated plasma concentrations.

Unexpectedly high absorption of the calcium salt was seen following oralabsorption although there was some variability in responses; meanestimated bioavailability including two lower responders was 25% and ashigh as 50% in some animals. TRI 50c was also well tolerated orallyalthough the dynamic responses were significantly less than those forthe calcium salt.

Example 22 TRI 50b Inhibition of Platelet Procoagulant Activity

Platelet pro-coagulant activity may be observed as the increase, in rateof activation of prothrombin by factor Xa in the presence of factor Vaupon the addition of platelets pretreated with thrombin, caused bythrombin alone, collagen alone or a mixture of thrombin and collagen.This property is due to an increase in anionic phospholipid on thesurface of the platelet with concomitant release of microvesicle fromthe surface. This is an essential physiological reaction and peoplewhose platelets have reduced ability to generate procoagulant activity(Scott syndrome) show an increased tendency for bleeding.

Method:

Washed platelets were treated with either 1.15 nM thrombin, 23 μg/mlcollagen or a mixture of both at the same concentration at 37° C. TRI50b was added either for 1 minute prior to the addition of activator orimmediately after the incubation with activator. Platelet procoagulantactivity was determined as described previously (Goodwin C A et al,Biochem J. 8(308):15-21, 1995).

TRI 50b proved to be a potent inhibitor of platelet procoagulantactivity with IC₅₀'s as summarised below:

Table 5: Influence of TRI 50b on the induction of platelet procoagulantactivity by various agonists:

TABLE 5 IC50 plus pre- IC50 without Fold acceleration incubationincubation Agonist without TRI 50b (nM) (nM) Thrombin 30 8 3000 Collagen45 200 300 Thrombin/Collagen 110 3 80

Table 5 records, for example, that when platelets were treated withthrombin they caused a 30-fold acceleration of the rate of activation ofprothrombin in comparison with control platelets. Treatment with TRI 50reduced such acceleration by half at the various TRI 50 concentrationlevels given. The significant potency of TRI 50 is evidenced by the factthat the IC₅₀ values are in the nanomolar range.

TRI 50b does not have an effect on ADP, collagen or epinephrine inducedaggregation of washed platelets.

Example 23 Rabbit Extracorporeal Shunt Model

Introduction

The technique describes an animal model in which a platelet richthrombus is produced. The activity of TRI 50b and heparin are compared.

The carotid artery and jugular vein of anaesthetised rabbits were usedto create an extracorporeal circuit containing a suspended foreignsurface (silk thread). Thrombus deposition is initiated by creation ofhigh sheer stress turbulent arterial blood flow, platelet activation,followed by coagulation in the presence of thrombogenic surfaces.Histopathological studies have shown that the thrombus is platelet rich.

Materials and Methods

Animals:

NZW rabbits (males 2.5-3.5 kg) were used. The animals were allowed foodand water up to the induction of anaesthesia.

Anaesthesia:

Animals were premedicated with fontanel/fluanisone (Hypnorm) 0.15 mltotal by intramuscular injection. General anaesthesia was induced withmethohexitone (10 mg/ml) to effect, followed by endotracheal intubation.Anaesthesia was maintained with isoflurane (1-2.0 %) carried inoxygen/nitrous oxide.

Surgical Preparation:

The animals were placed in dorsal recumbency and the ventral cervicalregion prepared for surgery. The left carotid artery and right jugularvein were exposed. The artery was cannulated with a large Portex®catheter (yellow gauge), cut to a suitable length. The vein wascannulated with a Silastic® catheter. The shunt comprised of a 5 cmlength of ‘auto analyser’ line (purple/white gauge). Joins to the shunton the arterial side were made with intermediate size Silastic® tubing.The shunt was filled with saline before exposure to the circulation. Theright femoral artery was cannulated for the measurement of bloodpressure.

Thread Preparation and Insertion:

The central section of the shunt contained a thread 3 centimetres inlength. This consisted of 000 gauge Gutterman sewing silk so as to givefour strands with a single knot at the end. (The knot section wasoutside the shunt).

Blood Flow

Blood flow velocity was determined by use of ‘Doppler’ probes (CrystalBiotech). A silastic probe was positioned over the carotid artery at thepoint of insertion of the arterial catheter. Flow was recorded on achart recorder using heat sensitive paper.

Results

TABLE 6 THROMBUS WEIGHT ANTITHROMBOTIC TREATMENT DOSE AFTER 20 minuterun ACTIVITY Control N/A 22.4 ± 2.2 mg (n = 5) TRI 50b  0 mg/kg iv 9.78± 1.9 mg (n = 5) Active  3.0 mg/kg iv 15.3 ± 2.2 mg (n = 5) ActiveHEPARIN 100 u/kg iv 22.9 ± 1.65 mg (n = 4)  Inactive 300 u/kg iv 10.5 ±1.4 mg (n = 4) Active (Severe bleeding)

Discussion

Table 6 shows that, under high arterial shear conditions, a TRI 50b doseof 3 mg/kg to 10 mg/kg iv significantly inhibits thrombus formationwithout bleeding, whereas a heparin dose within the normal clinicalrange for treating venous thrombosis (100 u/kg iv heparin) wasineffective. The higher dose of heparin, though active, caused severebleeding. These results, which show TRI 50b effectively inhibitingarterial thrombosis without causing bleeding, are consistent With TRI50b inhibiting platelet procoagulant activity. In contrast, the thrombininhibitor heparin, when administered at an approximately equi-effectivedose (in terms of inhibition of arterial thrombosis), produced thesevere bleeding normal when thrombin inhibitors are used to treatarterial thrombosis.

Example 24 Comparison of Bleeding Times

The aim of the study was to compare the bleeding times of heparin withTRI 50b in a suitable model. It is accepted that heparin is a poorinhibitor of platelet procoagulant activity (J. Biol. Chem.253(19):6908-16, 1978; Miletich J P, Jackson C M, Majerus P W1: J. Clin.Invest. 71(5):1383-91, 1983).

Bleeding times were determined in a rat tail bleeding model followingintravenous administration of heparin and TRI 50b. The doses employedwere chosen on the basis of their efficacy in the rat Wessler anddynamic models and were as follows:

TRI 50b: 5 and 10 mg/kg Heparin: 100 units/kg

Materials and Methods

Anaesthesia

Rats were anaesthetised with sodium pentabarbitone at 60 mg/kg (2.0ml/kg of 30 mg/ml solution by ip. injection). Supplemental anaestheticwas given ip. as required.

Surgical Preparation

A jugular vein was cannulated for the administration of test compound.The trachea was also cannulated with a suitable cannula and the animalsallowed to breathe ‘room air’ spontaneously.

Compound Administration

These were given in the appropriate vehicle at 1.0 ml/kg intravenously.Heparin was administered in saline, whilst TRI 50b was dissolved inethanol, and then the resultant solution added to water for injection (1part ethanol to 5 parts water).

Technique

Two minutes following compound administration the distal 2 mm of theanimal's tail was sectioned with a new scalpel blade and the tailimmersed in warm saline (37° C.) contained in a standard ‘universal’container, so that the blood stream was clearly visible. The bleedingtime recording was started immediately following transection until thecessation of blood flow from the tip of the tail. A period of 30 secondswas allowed after the blood flow from the tail had stopped to ensurethat bleeding did not re-commence, if bleeding did start again therecording time was continued for up to a maximum of 45 minutes.

Results

Table 7 gives a summary of the bleeding results and shows the increasesabove base line values.

TABLE 7 Summary table of bleeding results Bleeding time min Treatment(±SEM^(†)) Saline  5.1 ± 0.6 Heparin 100 u/kg iv >40* TRI 50b 5 mg/kg iv11.3 ± 1.2 TRI 50b 10 mg/kg 30.4 ± 5.2 iv *Severe bleeding in allanimals, with no cessation after 40 minutes. ^(†)SEM = standard error ofthe mean

Discussion

The results show that TRI 50b was superior to heparin (produced lessbleeding) at all doses. It should be noted that when 100 u/kg heparin iscompared with 5 mg/kg TRI 50b, heparin-treated animals bled moreextensively than those receiving TRI 50b; it was previously established(Example 23) that heparin at a dose of 100 u/kg is a less effectiveinhibitor of arterial thrombosis than TRI 50b at a dose of 3.0 mg/kg.Heparin is primarily a thrombin inhibitor and a poor inhibitor ofplatelet procoagulant activity; the results are therefore consistentwith TRI 50b exerting anti-coagulant activity by inhibition of plateletcoagulant activity in addition to thrombin inhibiting activity.

Example 25 TRI 50b as a Prodrug for TRI 50c: Pharmacokinetics andAbsorption

Materials and Methods

Animals

Rats, body weight circa 250-300 g were used. The animals were fastedonly on the day of use for the iv stage. Animals were fasted on thenight prior to study for the oral and intraduodenal studies, water wasallowed up to the time of anaesthesia.

TABLE 8 oral phase Treatment Dose mg/kg po n TRI 50b 20 mg/kg 2 TRI 50c20 mg/kg 2

TABLE 9 intraduodenal phase Treatment Dose mg/kg po n TRI 50b 20 mg/kg 3TRI 50c 20 mg/kg 3

Dose

Formulation (TRI 50b/TRI 50c)

These were dosed in a formulation prepared as follows: 48 mg/ml of TRI50b is dissolved in ethanol: PEG 300 (2:3 vol: vol). Just beforeadministration, 5 volumes of this solution is mixed with 3 volumes of 5%kollidon 17 8F.

Both compounds were dosed by oral gavage, or directly into the duodenum,at 20 mg/kg.

The compounds were dosed in a PEG/ethanol/kollidon formulation which wasprepared immediately before, as described immediately under the heading“Dose”: Stock 15.0 mg/ml. This was dosed at 1.33 ml/kg (equivalent to 30mg/kg).

Methods

Oral Gavage

Rats were dosed at 20 mg/kg. Approximately 30 minutes following dosingthe rats were anaesthetised.

Intraduodenal Administration

The compounds were instilled directly into the duodenum afteranaesthesia and surgical procedures had been completed.

Blood Sampling

Oral Phase

Blood (0.81 ml) was taken from the carotid cannula into (0.09 ml) of3.8% w/v tri sodium citrate following anaesthesia and surgery. The firstsamples were taken one-hour post dose. Then at, 1.5, 2, 4 hours postdose.

Intraduodenal Phase

Blood samples were taken: Pre dose, then at 0.25, 0.5, 0.75, 1.0, 2, 3and 4 hours post dosing.

Plasma

This was obtained by centrifugation (3000 RPM for 10 minutes) and storedat −20° C. prior to analysis.

Results

Pharmacokinetic Analysis

FIG. 5: oral phase clearance and kinetics following dosing with TRI 50bor its free acid (TRI 50c).

FIG. 6: oral phase clearance and kinetics following intraduodenal dosingwith TRI 50b or its free acid (TRI 50c).

Conclusion

When given by the intraduodenal route TRI 50b achieved a higherbioavailability (peak plasma concentration) than the free acid. The dataare consistent with TRI 50b being rapidly hydrolysed in plasma to TRI50c and with TRI 50c being the active principle.

Taken together with the data from examples 18 to 21, the results ofexamples 22 to 25 indicate that oral administration of TRI 50c as thecalcium salt will provide an excellent way to treat arterial thrombosisand/or venous thrombosis.

Example 26 Human Clinical Studies

In human clinical volunteer studies with doses of up to 2.5 mg/kg i.v.(dosages which significantly prolong the thrombin clotting time), TRI50b had no effect on Simplate bleeding time (i.e. bleeding time measuredusing a Simplate® bleeding time device).

It will be appreciated from the foregoing that boronic acid salts aredescribed that are useful for pharmaceutical purposes and which featureone or more of the following attributes: (1) improved amount of oralbioavailability; (2) improved consistency of oral bioavailability; (3)improved stability; and (4), in any event, not suggested by the priorart.

The selection of active ingredient for a pharmaceutical composition is acomplex task, which requires consideration not only of biologicalproperties (including bioavailability) but also of physicochemicalproperties desirable for processing, formulation and storage.Bioavailability itself is dependent on various factors, often includingin vivo stability, solvation properties and absorption properties, eachin turn potentially dependent on multiple physical, chemical and/orbiological behaviours.

The present disclosure includes the subject matter of the followingparagraphs:

1. An oral pharmaceutical formulation comprising a salt of apharmaceutically acceptable multivalent metal and an organoboronic aciddrug.

2. A formulation of paragraph 1 wherein the metal is a Group II or GroupIII metal or zinc.

3. A formulation of paragraph 1 or paragraph 2 wherein the metal isdivalent.

4. A formulation of paragraph 1 wherein the metal is calcium.

5. A formulation of paragraph 1 wherein the metal is magnesium.

6. A formulation of any of paragraphs 1 to 5 wherein the organoboronicacid is hydrophobic.

7. A formulation of any of paragraphs 1 to 6 wherein the organoboronicacid comprises a boropeptide or boropeptidomimetic.

8. A formulation of any of paragraphs 1 to 6 wherein the organoboronicacid is of the formula (I):

where:

R¹ is H or a non-charged side group;

R² is H or C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen orsulfur and optionally substituted by a substituent selected from halo,hydroxy and trifluoromethyl;

-   -   or R¹ and R² together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur;

R³ is the same as or different from R¹ provided that no more than one ofR¹ and R² is H, and is H or a non-charged side group;

R⁴ is H or C₁-C₁₃ hydrocarbyl optionally containing in-chain oxygen orsulfur and optionally substituted by a substituent selected from halo,hydroxy and trifluoromethyl;

-   -   or R³ and R⁴ together form a C₁-C₁₃ moiety which in combination        with N—CH forms a 4-6 membered ring and which is selected from        alkylene (whether branched or linear) and alkylene containing an        in-chain sulfur or linked to N—CH through a sulfur; and

R⁵ is X-E- wherein E is nothing or a hydrophobic moiety selected fromthe group consisting of amino acids (natural or unnatural) and peptidesof two or more amino acids (natural or unnatural) of which more thanhalf are hydrophobic and X is H or an amino-protecting group.

9. A formulation of paragraph 8 where R² and R⁴ are H, or R² is H and R³and R⁴ together form a said C₁-C₁₃ moiety.

10. A formulation of paragraph 8 or paragraph 9 wherein said hydrocarbyloptionally containing in-chain oxygen or sulfur is selected from thegroup consisting of alkyl; alkyl substituted by cycloalkyl, aryl orheteroaryl; cycloalkyl; aryl; and heteroaryl.

11. A formulation of any of paragraphs 8 to 10 wherein E is nothing.

12. A formulation of any of paragraphs 8 to 10 wherein E is ahydrophobic amino acid.

13. A formulation of any of paragraphs 1 to 6 wherein the organoboronicacid is of the formula (II):

wherein

R⁷ is X-E′- wherein X is hydrogen or an amino-protecting group and E′ isabsent or is a hydrophobic amino acid;

R⁸ is an optionally substituted moiety containing from 1 to 5 carbonatoms and selected from the group consisting of alkyl, alkoxy andalkoxyalkyl, the optional substituents being hydroxy and halogen (F, Cl,Br, I); and

aa^(h) is a hydrophobic amino acid, or is glycine N-substituted by aC₁-C₁₃ hydrocarbyl group optionally containing in-chain oxygen or sulfurand optionally substituted by a substituent selected from halo, hydroxyand trifluoromethyl.

14. A formulation of any of paragraphs 8 to 13 where X isR⁶—(CH₂)_(p)—C(O)—, R⁶—(CH₂)_(p)—S(O)₂—, R⁶—(CH₂)_(p)—NH—C(O)— orR⁶—(CH₂)_(p)—O—C(O)— wherein p is 0, 1, 2, 3, 4, 5 or 6 and R⁶ is H or a5 to 13-membered cyclic group optionally substituted by 1, 2 or 3substituents selected from halogen, amino, nitro, hydroxy, a C₅-C₆cyclic group, C₁-C₄ alkyl and C₁-C₄ alkyl containing, and/or linked tothe cyclic group through, an in-chain O, the aforesaid alkyl groupsoptionally being substituted by a substituent selected from halogen,amino, nitro, hydroxy and a C₅-C₆ cyclic group.

15. A formulation of paragraph 14 wherein said 5 to 13-membered cyclicgroup is aromatic or heteroaromatic.

16. A formulation of paragraph 15 wherein said 5 to 13-membered cyclicgroup is phenyl or a 6-membered heteroaromatic group.

17. A formulation of any of paragraphs 14 to 16 wherein X isR⁶—(CH₂)_(p)—C(O)— or R⁶—(CH₂)_(p)—O—C(O)— and p is 0 or 1.

18. A formulation of any of paragraphs 1 to 6 wherein the organoboronicacid is a serine protease inhibitor.

19. An oral pharmaceutical formulation comprising a salt of apharmaceutically acceptable multivalent metal and an organoboronic acidinhibitor of a coagulation serine protease.

20. A formulation of paragraph 19 wherein the organoboronic acid is apeptide boronic acid.

21. A formulation of paragraph 19 or paragraph 20 wherein theorganoboronic acid is a thrombin inhibitor.

22. A formulation of paragraph 21 wherein the thrombin inhibitor has aneutral thrombin S1-binding moiety linked to a hydrophobic thrombinS2/S3-binding moiety.

23. A formulation of paragraph 21 wherein the organoboronic acid is ofFormula (III):

wherein

Y comprises a moiety which, together with the fragment —CH(R⁹)—B(OH)₂,has affinity for the substrate binding site of thrombin; and

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages and in which the total number of oxygen and carbon atoms isfrom 3 to 6, or is —(CH₂)_(m)—W where m is from 2 to 5 and W is —OH orhalogen (F, Cl, Br or I).

24. A formulation of paragraph 23 wherein Y comprises an amino acidwhich binds to the S2 subsite of thrombin and is linked to—CH(R⁹)—B(OH)₂ by a peptide linkage, the amino acid being N-terminallylinked to a moiety which binds the S3 subsite of thrombin.

25. A formulation of paragraph 23 wherein Y is an optionallyN-terminally protected dipeptide residue which binds to the S3 and S2binding sites of thrombin and is linked to —CH(R⁹)—B(OH)₂ by a peptidelinkage, the peptide linkages in the acid optionally and independentlybeing N-substituted by a C₁-C₁₃ hydrocarbyl group optionally containingin-chain oxygen or sulfur and optionally substituted by a substituentselected from halo, hydroxy and trifluoromethyl.

26. A formulation of paragraph 25 wherein the N-terminal protectinggroup is a group X as defined in any of paragraphs 13 to 17 (other thanhydrogen).

27. A formulation of paragraph 25 or paragraph 26, wherein theorganoboronic acid has an N-terminal protecting group and unsubstitutedpeptide linkages.

28. An oral pharmaceutical formulation comprising a salt of apharmaceutically acceptable multivalent metal and a peptide boronic acidof formula (IV):

where:

X is H (to form NH₂) or an amino-protecting group;

aa¹ is an amino acid residue having a hydrocarbyl side chain containingno more than 20 carbon atoms and comprising at least one cyclic grouphaving up to 13 carbon atoms;

aa² is an imino acid residue having from 4 to 6 ring members;

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages and in which the total number of oxygen and carbon atoms isfrom 3 to 6, or is —(CH₂)_(m)—W where m is from 2 to 5 and W is —OH orhalogen (F, Cl, Br or I).

29. A formulation of paragraph 28 wherein aa¹ has a hydrocarbyl sidechain containing up to 13 C atoms.

30. A formulation of paragraph 28 wherein the cyclic group(s) of aa¹is/are aryl groups.

31. A formulation of paragraph 28 wherein the cyclic group(s) of aa¹is/are phenyl.

32. A formulation of paragraph 28 wherein aa¹ has a hydrocarbyl sidechain containing one or two cyclohydrocarbyl groups.

33. A formulation of paragraph 28 wherein aa¹ is Phe, Dpa or a wholly orpartially hydrogenated analogue thereof.

34. A formulation of paragraph 28 wherein aa¹ is selected from Dpa, Phe,Dcha and Cha.

35. A formulation of any of paragraphs 28 to 34 wherein aa¹ is ofR-configuration.

36. A formulation of paragraph 35 wherein aa¹ is (R)-Phe (that is,D-Phe) or (R)-Dpa (that is, D-Dpa).

37. A formulation of paragraph 35 wherein aa¹ is (R)-Phe.

38. A formulation of any of paragraphs 28 to 37 wherein aa² is a residueof an imino acid of formula (V)

where R¹¹ is —CH₂—, CH₂—CH₂—, —S—CH₂— or —CH₂—CH₂—CH₂—, which group,when the ring is 5- or 6- membered, is optionally substituted at one ormore —CH₂— groups by from 1 to 3 C₁-C₃ alkyl groups.

39. A formulation of paragraph 38 wherein aa² is of S-configuration.

40. A formulation of paragraph 38 wherein aa² is (S)-Pro.

41. A formulation of paragraph 28, wherein aa¹-aa² is (R)-Phe-(S)-Pro(that is, D-Phe-L-Pro).

42. A formulation of any of paragraphs 28 to 41 wherein R⁹ is2-bromoethyl, 2-chloroethyl, 2-methoxyethyl, 3-bromopropyl,3-chloropropyl or 3-methoxypropyl.

43. A formulation of any of paragraphs 28 to 41 wherein R⁹ is3-methoxypropyl.

44. A formulation of paragraph 28 wherein the peptide boronic acid is acompound of formula (IX):

X—(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂   (IX),

where X is as defined in paragraph 28 or paragraph 24.

45. A formulation of any of paragraphs 28 to 44 wherein X isR^(6′)—(CH₂)_(p)—C(O)— or R^(6′)—(CH₂)_(p)—O—C(O)—, where R^(6′) isphenyl or a 6-membered heteroaromatic group and p is 0 or 1.

46. A formulation of any of paragraphs 28 to 44 wherein X isbenzyloxycarbonyl.

47. A formulation of any of paragraphs 28 to 46 wherein the salt is adivalent metal salt of the peptide boronic acid.

48. A formulation of paragraph 47 wherein the metal is calcium.

49. A formulation of paragraph 47 wherein the metal is magnesium.

50. A formulation of any of paragraphs 28 to 46 wherein the metal is aGroup III metal salt of the peptide boronic acid.

51. A formulation of paragraph 50 wherein the metal is aluminium.

52. A formulation of paragraph 50 wherein the metal is gallium.

53. A formulation of any of paragraphs 1 to 52 which has a stoichiometryconsistent with the boronate groups in the formulation predominantlycarrying a single native charge.

54. A pharmaceutical composition adapted for oral administration andcomprising a calcium salt of the compound:

Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂.

55. A formulation of paragraph 54 wherein the salt is an acid salt.

56. A formulation of any of paragraphs 1 to 55 wherein the saltcomprises a boronate ion derived from the boronic acid and a counterionand wherein the salt consists essentially of a salt having a single typeof counterion.

57. A formulation of any of paragraphs 1 to 56 which is in the form of atablet or capsule.

58. A pharmaceutical formulation in oral dosage form comprising a saltas defined in any of paragraphs 1 to 56 and a pharmaceuticallyacceptable diluent, excipient or carrier.

59. A formulation of any of paragraphs 1 to 58 which is adapted torelease the salt or the product in the duodenum.

60. A formulation of paragraph 59 which is enterically coated.

61. A salt of a pharmaceutically acceptable multivalent (at leastdivalent) metal and an organoboronic acid drug (where the term “drug”embraces prodrugs), wherein the observed stoichiometry is consistentwith a predominant portion of the salt having a notional drug:metalstoichiometry of n:1, wherein n is the valency of the metal.

62. A salt of paragraph 61 wherein the observed stoichiometry isconsistent with the salt consisting essentially of a salt having anotional drug:metal stoichiometry of V:1.

63. A salt of paragraph 61 or paragraph 62 which is as further definedby any of paragraphs 1 to 53 or 55.

64. A salt of a pharmaceutically acceptable multivalent metal and anorganoboronic acid of Formula (III) as defined in any of paragraphs 23to 27.

65. A salt of a pharmaceutically acceptable multivalent metal and apeptide boronic acid of formula (IV):

where:

X is H (to form NH₂) or an amino-protecting group;

aa¹ is an amino acid residue having a hydrocarbyl side chain containingno more than 20 carbon atoms and comprising at least one cyclic grouphaving up to 13 carbon atoms;

aa² is an imino acid residue having from 4 to 6 ring members;

R⁹ is a straight chain alkyl group interrupted by one or more etherlinkages and in which the total number of oxygen and carbon atoms isfrom 3 to 6, or is —(CH₂)_(m)—W where m is from 2 to 5 and W is —H orhalogen (F, Cl, Br or I).

66. A salt of paragraph 65 which is as further defined by the featuresof any of paragraphs 29 to 52, or a permissible combination thereof.

67. A calcium salt of the compound Cbz-(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂.

68. A calcium salt of paragraph 67 which has an observed stoichiometry(compound:calcium) substantially of 2:1.

69. A composition of matter for use in preparing a salt of any ofparagraphs 65 to 68 comprising an alkali metal salt of an organoboronicacid as defined in any of paragraphs 28 to 46 or 54.

70. A sodium salt of a compound of Formula (IV) as defined in any ofparagraphs 23 to 46 or 54.

71. A potassium salt of a compound of Formula (IV) as defined in any ofparagraphs 23 to 46 or 54.

72. A salt of any of paragraphs 57 to 59 when in aqueous solution.

73. A method of inhibiting a coagulation serine protease in thetreatment of disease comprising orally administering to a mammal atherapeutically effective amount of a product selected from the groupconsisting of the formulations of any of paragraphs 19 to 60 and thesalts of any of paragraphs 64 to 68.

74. A method of paragraph 73 wherein the active agent is in aformulation adapted to release the active agent in the duodenum.

75. The use of a salt of any of paragraphs 64 to 68 for the manufactureof an oral medicament for treating, for example preventing, thrombosis.

76. A method of treating venous and/or arterial thrombosis byprophylaxis or therapy, comprising administering to a mammal sufferingfrom, or at risk of suffering from, venous and/or arterial thrombosis atherapeutically effective amount of a product selected from theformulation of any of paragraphs 23 to 60 and the salt of any ofparagraphs 64 to 68.

77. A method of paragraph 76 wherein the disease is an acute coronarysyndrome.

78. A method of paragraph 76 wherein the disease is acute myocardialinfarction.

79. A method of paragraph 76 wherein the disease is a venousthromboembolic event, selected from the group consisting of deep veinthrombosis and pulmonary embolism.

80. A method for preventing thrombosis in a haemodialysis circuit of apatient, comprising administering to the patient a therapeuticallyeffective amount of a product selected from the formulations of any ofparagraphs 23 to 60 and the salt of any of paragraphs 64 to 68.

81. A method for preventing a cardiovascular event in a patient with endstage renal disease, comprising administering to the patient atherapeutically effective amount of a product selected from theformulations of any of paragraphs 23 to 60 and the salt of any ofparagraphs 64 to 68.

82. A method for preventing venous thromboembolic events in a patientreceiving, or intended to receive, chemotherapy through an indwellingcatheter, comprising administering to the patient a therapeuticallyeffective amount of a product selected from the formulations of any ofparagraphs 23 to 60 and the salt of any of paragraphs 64 to 68.

83. A method for preventing thromboembolic events in a patientundergoing, or intended to undergo, a lower limb arterial reconstructiveprocedure, comprising administering to the patient a therapeuticallyeffective amount of a product selected from the formulation of any ofparagraphs 23 to 60 and the salt of any of paragraphs 64 to 68.

84. A method of inhibiting platelet procoagulant activity, comprisingadministering to a mammal at risk of, or suffering from, arterialthrombosis a therapeutically effective amount of a product selected fromthe formulations of any of paragraphs 23 to 60 and the salt of any ofparagraphs 64 to 68.

85. A method of paragraph 84 wherein the disease is an acute coronarysyndrome.

86. A method of treating by way of therapy or prophylaxis an arterialdisease selected from acute coronary syndromes, cerebrovascularthrombosis, peripheral arterial occlusion and arterial thrombosisresulting from atrial fibrillation, valvular heart disease,arterio-venous shunts, indwelling catheters or coronary stents,comprising administering to a mammal a therapeutically effective amountof a product selected from the formulations of any of paragraphs 23 to60 and the salt of any of paragraphs 64 to 68.

87. A method of paragraph 86 wherein the disease is an acute coronarysyndrome.

88. The use of a salt of any of paragraphs 64 to 68 for the manufactureof an oral medicament for a treatment recited in any of paragraphs 73and 75 to 87.

89. A pharmaceutical formulation comprising a combination of (i) a saltof any of paragraphs 64 to 68 and (ii) a further pharmaceutically activeagent.

90. A pharmaceutical formulation comprising a combination of (i) a saltof any of paragraphs 64 to 68 and (ii) another cardiovascular treatmentagent.

91. A formulation of paragraph 90 wherein the other cardiovasculartreatment agent comprises a lipid-lowering drug, a fibrate, niacin, astatin, a CETP inhibitor, a bile acid sequestrant, an anti-oxidant, aIIb/IIIa antagonist, an aldosterone inhibitor, an adenosine A2 receptorantagonist, an adenosine A3 receptor agonist, a beta-blocker,acetylsalicylic acid, a loop diuretic, an ace inhibitor, anantithrombotic agent with a different mechanism of action, anantiplatelet agent, a thromboxane receptor and/or synthetase inhibitor,a fibrinogen receptor antagonist, a prostacyclin mimetic, aphosphodiesterase inhibitor, an ADP-receptor (P₂ T) antagonist, athrombolytic, a cardioprotectant or a COX-2 inhibitor.

92. The use of a salt as defined in any of paragraphs 23 to 60 for themanufacture of a medicament for treating, for example preventing, acardiovascular disorder in co-administration with another cardiovasculartreatment agent.

93. A product comprising an adduct of a compound of Formula (IX) asdefined in paragraph 44 and diethanolamine.

94. A composition of matter comprising:

-   -   (i) a species of formula (X)

wherein X is H or an amino protecting group, the boron atom isoptionally coordinated additionally with a nitrogen atom, and thevalency status of the terminal oxygens is open (they may be attached toa second covalent bond, be ionised as —O⁻, or have some other, forexample intermediate, status); and, in bonding association therewith

-   -   (ii) a species of formula (XI)

wherein the valency status of the nitrogen atom and the two oxygen atomsis open.

95. A composition of paragraph 94, wherein the terminal oxygen atoms ofthe species of formula (X) and the oxygen atoms of the species offormula (XI) are the same oxygen atoms, i.e. the species of formula (XI)forms a diol ester with the species of formula (X).

96. A medicament comprising a salt of a pharmaceutically acceptabledivalent metal and an organoboronic acid which is a selective thrombininhibitor and has a neutral thrombin S1 subsite-binding moiety.

97. A medicament of paragraph 96 wherein the selective thrombininhibitor is an organoboronic acid of Formula (III) as defined in any ofparagraphs 23 to 27.

98. A medicament of paragraph 96 or paragraph 97 wherein the selectivethrombin inhibitor has a Ki for thrombin of about 100 nM or less.

99. A medicament of paragraph 98 wherein the selective thrombininhibitor has a Ki for thrombin of about 20 nM or less.

100. A method of stabilising an organoboronic acid, comprising providingit in the form of a multivalent salt thereof.

101. A method of formulating an organoboronic acid drug to increase thestability of the drug species, comprising formulating the acid in theform of an acid salt thereof with a multivalent metal.

1. A pharmaceutical formulation adapted for oral administration andcomprising a) a first species selected from the group consisting of aboronic acid of formula (III), said acid when in the form of a boronateion thereof, an equilibrium form of said boronic acid and of saidboronate ion, and combinations thereof:

wherein Y comprises a moiety which, together with the aminoboronic acidresidue —NHCH(R⁹)—B(OH)₂, has affinity for the substrate binding site ofthrombin; and R⁹ is a straight chain alkyl group interrupted by one ormore ether linkages and in which the total number of oxygen and carbonatoms is 3, 4, 5 or 6 or R⁹ is —(CH₂)_(m)—W where m is from 2, 3, 4 or 5and W is —OH or halogen; and (b) a second species selected from thegroup consisting of multivalent metal ions.
 2. The formulation of claim1 wherein R⁹ is an alkoxyalkyl group.
 3. The formulation of claim 1wherein Y comprises an amino acid which binds to the S2 subsite ofthrombin and is linked to —CH(R⁹)—B(OH)₂ by a peptide linkage, the aminoacid being N-terminally linked to a moiety which binds the S3 subsite ofthrombin.
 4. The formulation of claim 1 wherein Y comprises a dipeptideresidue which binds to the S3 and S2 binding sites of thrombin and islinked to —CH(R⁹)—B(OH)₂ by a peptide linkage.
 5. The formulation ofclaim 1 wherein the boronic acid or boronate ion has a Ki for thrombinof about 100 nM or less.
 6. The formulation of claim 4 wherein the Ydipeptide is N-terminally protected or N-terminally unprotected, and thepeptide linkages in the dipeptide are unsubstituted or independentlyN-substituted by a C₁-C₁₃ hydrocarbyl, wherein the C₁-C₁₃ hydrocarbylcontains no heteroatoms or at least one in-chain or in-ring nitrogen,oxygen or sulfur atom, and the C₁-C₁₃ hydrocarbyl is unsubstituted orsubstituted by a substituent selected from halo, hydroxy ortrifluoromethyl.
 7. The formulation of claim 1 wherein the multivalentmetal comprises calcium, magnesium or zinc.
 8. The formulation of claim7 which has an observed stoichiometry which would be consistent with thefirst species being a boronate ion carrying a single negative charge. 9.The formulation of claim 2 wherein the second species is calcium ormagnesium ions and the observed stoichiometry of the first species tothe second species is about 2 to
 1. 10. The formulation of claim 1wherein the boronic acid is a peptide boronic acid of formula (IV):

where: X is H or an amino-protecting group; aa¹ is an amino acid havinga hydrocarbyl side chain containing no more than 20 carbon atoms andcomprising at least one cyclic group having up to 13 carbon atoms; aa²is an imino acid having from 4 to 6 ring members; R¹ is a group of theformula —(CH₂)_(s)-Z, where s is 2, 3 or 4 and Z is —OH, —OMe, —OEt orhalogen.
 11. The formulation of claim 11 wherein aa¹ is selected fromPhe, Dpa and wholly or partially hydrogenated analogues thereof.
 12. Theformulation of claim 11 wherein aa¹ is of R-configuration.
 13. Theformulation of claim 10 wherein aa² is a residue of an imino acid offormula (V)

where R¹¹ is —CH₂—, —CH₂—CH₂—, —S—CH₂—, —S—C(CH₃)₂— or —CH₂—CH₂—CH₂—,and, when the formula (V) ring is 5- or 6-membered, the formula (V) ringis unsubstituted or is substituted at one or more —CH₂— groups by from 1to 3 C₁-C₃ alkyl groups.
 14. The formulation of claim 13 wherein aa² isof S-configuration.
 15. The formulation of claim 14 wherein aa¹ is of(R)-configuration and the fragment —NH—CH(R¹)—B(OH)₂ is of(R)-configuration.
 16. The formulation of claim 11, wherein aa¹-aa² is(R)-Phe-(S)-Pro and the fragment —NH—CH(R¹)—B(OH)₂ is ofR-configuration.
 17. The formulation of claim 10 wherein R¹ ismethoxypropyl, aa² is of (S)-configuration, aa¹ is of (R)-configurationand the fragment —NH—CH(R¹)—B(OH)₂ is of (R)-configuration.
 18. Theformulation of claim 17 wherein the second species consists essentiallyof a calcium ion or a magnesium ion.
 19. The formulation of claim 10wherein the boronic acid is of formula (IX):X—(R)-Phe-(S)-Pro-(R)-Mpg-B(OH)₂   (IX), wherein X is R⁶(CH₂)_(p)—C(O)—,R⁶—(CH₂)_(p)—S(O)₂—, R⁶—(CH₂)_(p)—NH—C(O)— or R⁶—(CH₂)_(p)—O—C(O)—wherein p is 0, 1, 2, 3, 4, 5 or 6 and R⁶ is H or a 5 to 13-memberedcyclic group which is unsubstituted or substituted by 1, 2 or 3substituents selected from halogen; amino; nitro; hydroxy; a C₅-C₆cyclic group; C₁-C₄ alkyl and C₁-C₄ alkyl containing, or linked to thecyclic group through, an in-chain O atom, the aforesaid alkyl groupsoptionally being substituted by a substituent selected from halogen,amino, nitro, hydroxy and a C₅-C₆ cyclic group.
 20. The formulation ofclaim 10 wherein the second species is selected from a divalent metalion.
 21. The formulation of claim 1 which further comprises apharmaceutically acceptable diluent, excipient or carrier.
 22. Theformulation of claim 1 wherein the first species comprise anhydridespecies
 23. The formulation of claim 17 wherein the first speciescomprise anhydride species.
 24. The formulation of claim 1 wherein theboronic acid is Cbz-(R)-Phe-(S)-Pro-(R)-boroMpg-OH, the first speciescomprise anhydride species and the second species consists essentiallyof calcium ions, and wherein the formulation further includes apharmaceutically acceptable diluent, excipient or carrier.
 25. Amedicament adapted for oral administration and comprising atherapeutically effective amount of a multivalent metal salt of aboronic acid which is a selective thrombin inhibitor and has a neutralaminoboronic acid residue capable of binding to the thrombin S1 subsitelinked through a peptide linkage to a hydrophobic moiety capable ofbinding to the thrombin S2 and S3 subsites, the salt comprising a cationhaving a valency n and having an observed stoichiometry consistent witha notional stoichiometry (boronic acid:cation) of n:1.
 26. A medicamentof claim 25 which is in solid dosage form.
 27. A medicament of claim 26wherein the boronic acid has a Ki for thrombin of about 100 nM or less.28. A method for preventing thrombosis in a haemodialysis circuit of apatient, for preventing a cardiovascular event in a patient with endstage renal disease, for preventing venous thromboembolic events in apatient receiving chemotherapy through an indwelling catheter, forpreventing thromboembolic events in a patient undergoing a lower limbarterial reconstructive procedure, or for treating by way of therapy orprophylaxis an arterial disease selected from acute coronary syndromes,cerebrovascular thrombosis, peripheral arterial occlusion and arterialthrombosis resulting from atrial fibrillation, valvular heart disease,arterio-venous shunts, indwelling catheters or coronary stents, themethod comprising parenterally administering to a mammal atherapeutically effective amount of a salt of a pharmaceuticallyacceptable multivalent metal and a peptide boronic acid of formula (IV):

where: X is H or an amino-protecting group; aa¹ is an amino acid havinga hydrocarbyl side chain containing no more than 20 carbon atoms andcomprising at least one cyclic group having up to 13 carbon atoms; aa²is an imino acid having from 4 to 6 ring members; R¹ is a group of theformula —(CH₂)_(s)-Z, where s is 2, 3 or 4 and Z is —OH, —OMe, —OEt orhalogen.
 29. The formulation of claim 1 wherein the boronic acid isCbz-(R)-Phe-(S)-Pro-(R)-boroMpg-OH.
 30. The formulation of claim 1wherein the second species is calcium.